Methylotrophic yeast producing mammalian type sugar chain

ABSTRACT

This invention is to provide a process for producing a glycoprotein comprising a mammalian type sugar chain, characterized in that the process comprises introducing an α-1,2-mannosidase gene into a methylotrophic yeast having a mutation of a sugar chain biosynthesizing enzyme gene, so that the α-1,2-mannosidase gene is expressed under the control of a potent promoter in the yeast; culturing in a medium the methylotrophic yeast cells with a heterologous gene transferred thereinto; and obtaining the glycoprotein comprising a mammalian type sugar chain from the culture. Using the newly created methylotrophic yeast having a sugar chain mutation, a neutral sugar chain identical with a high mannose type sugar chain produced by mammalian cells such as human cells, or a glycoprotein comprising such a neutral sugar chain, can be produced in a large amount at a high purity. By introducing a mammalian type sugar chain biosynthesizing gene into the above-described mutant, a mammalian type sugar chain, such as a hybrid or complex, or a protein comprising a mammalian type sugar chain can be efficiently produced.

TECHNICAL FIELD

The present invention provides a process for mass production ofnon-antigenic mammalian type glycoproteins comprising a sugar chainstructure at their asparagine residues using a methylotrophic yeastwherein the sugar chain structure is identical to that produced bymammalian cells. More specifically, the present invention relates to anovel mutant yeast capable of producing a glycoprotein comprising amammalian type sugar chain, which is created by introducing anα-1,2-mannosidase gene into a methylotrophic yeast having a mutation ofsugar chain biosynthesizing enzyme genes, so that the α-1,2-mannosidasegene is highly expressed under the control of a potent promoter in theyeast, and the α-1,2-mannosidase exists in the endoplasmic reticulum(ER); and to a process for producing a glycoprotein comprising amammalian type sugar chain wherein the process comprises culturing themethylotrophic yeast cells with a heterologous gene transferredthereinto in a medium and obtaining the glycoprotein comprisingmammalian type sugar chains from the culture.

BACKGROUND OF THE INVENTION

Yeast has been intensively studied as a host for production of foreigngenes since establishment of yeast transformation systems. The use of ayeast for production of foreign proteins involves advantages in thatmolecular-genetic manipulation and culture of yeasts are as easy asthose of prokaryotic organisms, and that yeasts bear eukaryotic typefunctions to allow post-translational modifications of proteins such asglycosylation. However, since production of proteins using Saccharomycescerevisiae is low with exception of some successes, protein productionsystems using yeasts other than Saccharomyces cerevisiae have beendeveloped, including systems using, for example, Shizosaccharomycespombe, Kluyveromyces lactis, methylotrophic yeasts, or the like.

A methylotrophic yeast (or methanol-utilizing yeast), which can grow onmethanol as a single carbon source, has been developed as a host forproduction of foreign proteins (K. Wolf (ed.) “Non Conventional Yeastsin Biotechnology” (1996)). This is because methods of culturing yeastshave been established in industrial scale and because the yeast has apotent promoter controlled by methanol. At that time when amethylotrophic yeast was discovered, the use thereof as SCP (Single CellProtein) was studied and, as a result, a high-density culture techniqueat a dry cell weight of 100 g/L or more was established in aninexpensive culture medium, which contains minerals, trace elements,biotin, and carbon sources.

Researches on elucidation of a C1 compound-metabolic pathway, as well ason application of C1 compounds, revealed that a group of enzymesrequired for the methanol metabolism was strictly regulated by carbonsources. The methanol metabolism in a methanol-utilizing yeast generatesformaldehyde and hydrogen peroxide from methanol and oxygen by alcoholoxidase in the first reaction. The generated hydrogen peroxide isdecomposed into water and oxygen by catalase, while formaldehyde isoxidized to carbon dioxide by actions of formaldehyde dehydrogenase,S-formylglutathione hydrolase, and alcohol oxidase, and NADH generatedduring the oxidation is utilized as an energy source of the cell. At thesame time, formaldehyde is condensed with xylulose-5-phosphate bydihydroxyacetone synthase, then converted intoglyceraldehyde-3-phosphate and dihydroxyacetone, which subsequentlyenter the pentose phosphate pathway and serve as cell components.

Alcohol oxidase, dihydroxyacetone synthase, and formate dehydrogenaseare not detected in the cell when it is cultured in the presence ofglucose, but they are induced in the cell cultured in methanol, so thatthe amount of them is dozens of percentage of the total inner cellprotein. Since the production of these enzymes is controlled at atranscription level, inducible expression of a foreign gene of interestis enabled under the regulation of promoters of the genes which encodethe enzymes. The foreign gene expression system using a promoter for amethanol metabolizing enzyme gene has been estimated so highly amongyeast expression systems due to its efficient production, with anexample in which the expression amount of a foreign gene was dozens ofpercentage of the total protein in cell or several g/L culture medium insecretion.

To date, four types of the transformation and foreign gene expressionsystems have been established in the methylotrophic yeasts: Candidaboidinii, Hansenula polymorpha, Pichia pastoris and Pichia methanolica.Differences are recognized among the expression systems in terms ofcodon usage, expression regulation, and integration of expressionplasmid, which provide characteristics of each expression system.

In the meantime, it is known that naturally occurring proteins areclassified into two types, i.e., the one being a simple proteincomprising amino acids alone, the other being a complex proteincomprising sugar chains, lipids, phosphates or the like attachedthereto, and that most of cytokines are glycoproteins. Recently, besidesconventional analyses with lectin, new analyses using HPLC, NMR orFAB-MAS have been developed in analyzing sugar chain structures, bywhich new sugar chain structures of a glycoprotein have been foundsuccessively. On the other hand, studies on functional analysis of sugarchains lead to elucidation of the fact that the sugar chain plays animportant role in lots of bio-mechanisms, such as intercellularrecognition, molecular recognition, keeping of protein structures,contribution to protein activity, in vivo clearance, secretion,localization, etc.

For example, it has been revealed that erythropoietin (EPO), tissueplasminogen activator (TPA) or the like did not exhibit its inherentbioactivity when the sugar chains are removed (Akira Kobata,Tanpakushitsu-Kakusan-Koso, 36, 775-788 (1991)). Importance of sugarchains has been pointed out in erythropoietin, which was the firstglycoprotein medicament in history produced by transgenic animal cellsas the host. Specifically, the sugar chains of erythropoietin act ininhibitory manner against binding to receptor, whereas they have adecisive contribution to keeping of active structures and to improvementin in vivo pharmacokinetics, and are totally essential for expression ofthe pharmacological activity (Takeuchi and Kobata, Glycobiology, 1,337-346 (1991)). Furthermore, high correlation between the structure,type and number of branches (i.e., the number of branches formed byGlcNAc attached to Man3GlcNAc2) of sugar chains and the pharmacologicaleffect of erthropoietin has been found (Takeuchi et al., Proc. Natl.Acad. Sci. USA, 86, 7819-7822 (1989)). It was reported that a main causeof this phenomenon was that erythropoietin with immature branchstructure is prone to occur its rapid clearance from the kidney,resulting in a shorter retention time in the body (Misaizu et al.,Blood, 86, 4097-4104 (1995)). Another similar example is observed inserum glycoproteins including fetuin. That is, it was found that whenremoval of sialic acid at the end of a sugar chain leads to exposure ofgalactose, the galactose is recognized by lectin on the surface of livercells, whereby the serum glycoprotein disappears promptly from the blood(Ashwell and Harford, Annu. Rev. Biochem., 51, 531-554 (1982); Morell etal., J. Biol. Chem., 243, 155-159 (1968)).

Glycoprotein sugar chains are largely classified into Asn-linked(N-linked), mucin type, O-GlcNAc type, GPI-anchored type, andproteoglycan type (Makoto Takeuchi, Glycobiology Series 5,Glycotechnology; edited by Akira Kibata and Senichiro Hakomori,Katsutaka Nagai, Kodansha Scientific, 191-208 (1994)), each of which hasan intrinsic biosynthesis pathway and serves for individualphysiological functions. Of them, for the biosynthesis pathway ofAsn-linked sugar chains, there are many findings and detailed analyses.

Biosynthesis of Asn-linked sugar chains starts with synthesis of aprecursor comprising N-acetylglucosamine, mannose and glucose on a lipidcarrier intermediate, which precursor is converted to a specificsequence (Asn-X-Ser or -Thr) of a glycoprotein in the endoplasmicreticulum (ER). It is then subjected to processing (i.e., cleavage ofglucose and specific mannose residues) to synthesize an M8 high-mannosetype sugar chain comprising eight mannose residues and twoN-acetylglucosamine residues (Man8GlcNAc2). The protein including highmannose type sugar chains is transported to the Golgi apparatus whichundergoes a variety of modifications significantly different betweenyeast and mammal (Gemmill, T. R., Trimble, R. B., Biochim. Biophys.Acta., 1426, 227 (1999)).

In mammalian cells, in many cases, α-mannosidase I (α-1,2-mannosidase),an exomannosidase which cleaves an α-1,2-mannoside linkage, acts on highmannose type sugar chains to cut off several mannose residues. The sugarchain (Man5-8GlcNAc2) generated in this process is a sugar chainreferred to as a high mannose type. N-acetylglucosaminyl transferase(GnT) I acts on an M5 high mannose type sugar chain (Man5GlcNAc2) fromwhich three mannose residues have been cut off, to transfer oneN-acetylglucosamine residue to the sugar chain, resulting in formationof a sugar chain comprising GlcNAcMan5GlcNAc2. The thus formed sugarchain is referred to as a hybrid type. Further, when α-mannosidase IIand GnT II act, the sugar chain structure GlcNAc2Man3GlcNAc2, referredto as a complex type, is formed. Thereafter, a variety of mammalian typesugar chains are formed through the action of a group of ten-oddglycosyltransferase enzymes, by which addition of N-acetylglucosamine,galactose, sialic acid, etc. occurs (FIG. 1).

Accordingly, the mammalian type sugar chain as defined in thisapplication means an N-linked (or Asn-linked) sugar chain present inmammals, which is generated in the sugar chain biosynthesis process ofmammals. Specifically, they include an M8 high mannose type sugar chainrepresented by Man8GlcNAc2; an M5, M6 or M7 high mannose type sugarchain represented by Man5GlcNAc2, Man6GlcNAc2 or Man7GlcNAc2,respectively, generated from Man8GlcNAc by action of α-mannosidase I; ahybrid type sugar chain represented by GlcNAcMan5GlcNAc2 generated fromMan5GlcNAc2 by action of GlcNAc transferase-I (GnT-I); a double-strandedcomplex type sugar chain represented by GlcNAc2Man3GlcNAc2 generatedfrom GlcNAcMan5GlcNAc2 by action of α-mannosidase-I and GlcNActransferase-II (GnT-II); and a double-stranded complex type sugar chainrepresented by Gal2GlcNAc2Man3GlcNAc2 generated from GlcNAc2Man3GlcNAc2by action of galactosyl transferase (GalT).

In mammals, any of high mannose type, hybrid type and complex type sugarchains can be found. In one case, sugar chains to be attached aredifferent depending on a protein, or in another, different types ofsugar chains are attached within a protein. These sugar chains exhibitimportant functions, such as biosynthesis of glycoproteins, sortingwithin a cell, concealment of antigenicity, in vivo stability,organ-targeting properties, and the like, depending on the type or classof sugar chains attached to a glycoprotein (Tamao Endo, Tosa Kogaku(Sugar chain engineering), Sangyo Chosakai, 64-72 (1992)).

On the other hand, in yeast a mannan-type sugar chain (outer sugarchain) is produced, in which several to 100 or more mannose residues areattached to M8 high mannose type sugar chain. For example, thebiosynthesis of outer sugar chains in Saccharomyces cerevisiae known asbaker's yeast or laboratory yeast is considered to proceed along apathway as shown in FIG. 2 (Ballou et al., Proc. Natl. Acad. Sci. USA,87, 3368-3372 (1990)). That is, a reaction for initiating elongationbegins in which a mannose is first attached to M8 high mannose typesugar chain through α-1,6 linkage (FIG. 2, Reaction I, B). The enzymeperforming this reaction is clarified as a protein encoded by OCH1 gene(Nakayama et al., EMBO J., 11, 2511-2519 (1992)). Further, sequentialelongation of mannose by α-1,6-linkage reaction (FIG. 2, II), forms apoly α-1,6-mannose linkage being the backbone of an outer sugar chain(FIG. 2, E). The α-1,6-mannose linkage sometimes contains a branch ofα-1,2-linked mannose (FIG. 2: C, F, H), and additionally, α-1,3-linkedmannose is attached to the end of the branched α-1,2-linked mannosechain (FIG. 2: D, G, H, I). The addition of the α-1,3-linked mannose iscaused by a MNN1 gene product (Nakanishi-Shindo et al., J. Biol. Chem.,268, 26338-26345 (1993)). Formation of an acidic sugar chain, in whichmannose-1-phosphate has been attached to high mannose type sugar chainmoieties and outer chain moieties, is known as well (FIG. 2, *; apossible phosphorylation site corresponding to * in the above formula(I)). This reaction was found to be caused by a protein encoded by MNN6gene (Wang et al., J. Biol. Chem., 272, 18117-18124 (1997)). Further, agene (MNN4) coding for a protein positively regulating the transferreaction was clarified (Odani et al., Glycobiology, 6, 805-810 (1996);Odani et al., FEBS Letters, 420, 186-190 (1997)).

Production of substances using microorganisms including yeast has someadvantages as mentioned above, such as low production costs andutilizing culture technology developed as fermentation engineering, ascompared with the production of substances using animal cells. There isa problem, however, that microorganisms cannot attach sugar chains withthe same structure as human glycoprotein. Specifically, glycoproteinsfrom cells of an animal including human have a variety of mucin typesugar chains in addition to three kinds of Asn-linked sugar chains,i.e., complex type, hybrid type and high mannose type as shown in FIG.1, while the Asn-linked sugar chain whose attachment is observed even inbaker's yeast (Saccharomyces cerevisiae), is only a high mannose type,and a mucin type is attached only to a sugar chain mainly composed ofmannose.

Such sugar chains of yeast may produce a heterogeneous protein productresulting in difficulties in purification of the protein or in reductionof specific activity (Bekkers et al., Biochim. Biophys. Acta, 1089,345-351 (1991)). Furthermore, since the structure of the sugar chainssignificantly differ, glycoproteins produced by yeast may not have thesame detectable biological activity as those of the mammalian origin, orhave strong immunogenicity to a mammal, etc. Thus, yeast is unsuitableas a host for producing useful glycoproteins from mammalian origin, andin general microorganisms are not suitable for DNA recombinantproduction of a glycoprotein, such as erythropoietin as described above,in which sugar chain has an important function. Indeed, for productionof erythropoietin, Chinese hamster ovary (CHO) cells are used.

Thus, it is expected that the sugar chain of a glycoprotein not only hasa complicated structure but also plays an important role in expressionof biological activity. However, since the correlation of the structureof sugar chain with biological activity is not necessarily clear,development of the technology, which enables to freely modify or controlthe structure (the type of sugar, a linking position, chain length,etc.) of a sugar chain attached to a protein moiety, is needed. Whendeveloping a glycoprotein especially as medicament, the structure andfunction analyses of the glycoprotein become important. Under thesecircumstances, the development of yeast, which can produce aglycoprotein with biological activity equivalent to that of themammalian origin, i.e., a glycoprotein comprising a mammalian type sugarchain, is desired by the academic society and the industrial world.

In order to produce a mammalian type sugar chain using yeast, it isimportant to prepare a mutant having the sugar chain biosynthesissystem, which does not comprise a reaction as mentioned above ofattaching a lot of mannose residues to modify the glycoprotein sugarchain as seen particularly in yeast; in which no outer sugar chains areattached; and the synthesis of sugar chains generates M5 high mannosetype sugar chain. Subsequently, M8 high mannose type sugar chain, aprecursor for this mammalian type sugar chain, might be produced byintroducing biosynthetic genes for the mammalian type sugar chain intothe mutant yeast.

To obtain a glycoprotein lacking outer sugar chains, use of a mutantstrain deficient in enzymes for producing outer sugar chains in yeast,particularly a mutant of Saccharomyces cerevisiae, has been studied sofar. Methods to obtain such a deficient mutant strain include obtaininga gene mutant by chemicals, ultraviolet irradiation or natural mutation,or obtaining it by artificial disruption of a target gene.

As to the former methods, there are many reports thereon. For example,mnn2 mutant is defective in the step of branching which causes α-1,2linkage from the α-1,6 backbone of an outer sugar chain, and mnn1 mutantis defective in the step of producing α-1,3-linked mannose at the end ofthe branch. However, these mutants do not have defects in α-1,6 mannoselinkage as the backbone of outer sugar chains and so they produce a longouter sugar chain in length. Mutants like mnn7, 8, 9, 10 mutants havebeen isolated as mutants having only about 4 to 15 molecules of the α-1,6 mannose linkage. In these mutants, the outer sugar chains are merelyshortened, but the elongation of high mannose type sugar chains does notstop (Ballou et al., J. Biol. Chem., 255, 5986-5991 (1980); Ballou etal., J. Biol. Chem., 264, 11857-11864 (1989)). Defects in the additionof outer sugar chains are also observed in, for example, secretionmutants such as sec18 in which the transportation of a protein fromendoplasmic reticulum to Golgi apparatus is temperature-sensitive.However, in a sec mutant, since the secretion of a protein itself isinhibited at a high temperature, the sec mutant is not suitable forsecretion and production of glycoproteins.

Accordingly, since these mutants cannot completely biosynthesize thehigh mannose type sugar chain of interest, they are consideredunsuitable as host yeast for producing a mammalian type sugar chain.

On the other hand, as to the latter, the deficient mutant strain inwhich a plurality of target genes have been disrupted can be establishedby development of genetic engineering techniques in recent years.Specifically, through in vitro operation, a target gene DNA on plasmidis first fragmentated or partially deleted, and an adequate selectablemarker DNA is inserted at the fragmented or deleted site to prepare aconstruct in which the selectable marker is sandwiched between upstreamand downstream regions of the target gene. Subsequently, the linear DNAhaving this structure is transferred into a yeast cell to cause twohomologous recombinations at portions homologous between both ends ofthe introduced fragment and the target gene on chromosome, therebysubstituting the target gene with a DNA construct in which theselectable marker has been sandwiched (Rothstein, Methods Enzymol., 101,202-211 (1983)).

Molecular cloning of a yeast strain deficient in outer sugar chain hasalready been described by Jigami et al. in Japanese Patent Publication(Kokai) No. 6-277086A (1994) and No. 9-266792A (1997). Jigami et al.succeeded in cloning of the S. cerevisiae OCH1 gene (which expressesα-1,6-mannosyl transferase), the OCH1 enzyme being assumed to be a keyenzyme for elongation of the α-1,6 linked mannose. The glycoprotein ofthe OCH1 gene knockout mutant (Δoch1) had three types of attached sugarchains, i.e., Man8GlcNAc2, Man9GlcNAc2 and Man10GlcNAc2. Of them, theMan8GlcNAc2 chain had the same structure (i.e., the structure shown inFIG. 2A) as the ER core sugar chain which was common between S.cerevisiae and mammalian cell, while the Man9GlcNAc2 and Man10GlcNAc2chains had a structure where α-1,3-linked mannose was attached to thisER core sugar chain [Nakanish-Shindo, Y., Nakayama, K., Tanaka, A.,Toda, Y. and Jigami, Y., (1994), J. Biol. Chem.]. Furthermore, a S.cerevisiae host which can attach only the Man8GlcNAc2 chain having thesame structure as the ER core sugar chain, which structure is commonbetween S. cerevisiae and mammalian cell, was successfully produced bypreparing a Δoch1mnn1 dual mutant and inhibiting the α-1,3-linkedmannose transfer at the end. It is supposed that this Δoch1mnn1 doublemutant serves as a host useful in case where the mammalian glycoprotein,which has a high mannose type sugar chain, is produced by DNArecombinant technology [Yoshifumi Jigami (1994)Tanpakushitsu-Kakusan-Koso, 39, 657].

It was found, however, that sugar chains of the glycoprotein produced bythe double mutant (Δoch1mnn1) described in Japanese Patent Publication(Kokai) No. 6-277086 (1994) comprised acidic sugar chains containing aphosphate residue. This acidic sugar chain has a structure which is notpresent in sugar chains of mammals including human, and it is likely tobe recognized as a foreign substance in mammal, thereby exhibitingantigenicity (Ballou, Methods Enzymol., 185, 440-470 (1990)). Hence, aquadruple mutant (as described in Japanese Patent Publication (Kokai)No. 9-266792A (1997)) was constructed in which the functions of a genefor positively regulating the transfer of mannose-1-phosphate (MNN4) andof a mannose transferase gene for performing the elongation reaction foran O-linked sugar chain (KRE2) have been disrupted. It was revealed thatthe sugar chain of a glycoprotein produced by the yeast strain describedtherein had the M8 high mannose type sugar chain of interest. It wasfurther found that a strain in which Aspergillus saitoi-derivedα-1,2-mannosidase gene is transferred to a yeast cell where a geneinvolved in the particular sugar chain biosynthesis system of yeast hasbeen disrupted, had a high mannose type sugar chain (Man5-8GlcNAc2) inwhich one to several mannose residues were cleaved (Chiba et al., J.Biol. Chem., 273, 26298-26304 (1998)). Furthermore, they attemptedproduction of a mammalian type glycoprotein in yeast by transfer of agene involved in the mammalian sugar chain biosynthesis system into thisprepared strain (PCT/JP 00/05474). However, despite that anα-1,2-mannosidase gene was expressed using a promoter forglyceraldehyde-3-phosphate dehydrogenase gene which is considered to bethe highest in the expression amount as a constitutive expressionpromoter according to the disclosure, the conversion efficiency toMan5GlcNAc2 by carboxypeptidaseY (CPY) in the cell wall-derivedmannoprotein is as low as 10-30% and so it is hard to say that itsapplication to various glycoproteins is sufficiently prospective,although the rate of conversion to a high mannose type sugar chain(Man5GlcNAc2) was almost 100% in FGF as a foreign protein.

Separately, Schwientek et al. reported on the expression of the activityof human β-1,4-galactosyl transferase gene in S. cerevisiae in 1994[Schwientek, T. and Ernst, J. F., Gene, 145, 299 (1994)]. Similarly,Krezdrn et al. achieved the expression of the activity of humanβ-1,4-galactosyl transferase gene and α-2,6-sialyl transferase in S.cerevisiae [Krezdrn, C. H. et al., Eur.J.Biochem.220, 809 (1994)].

However, when these findings are tried to be applied to other yeast,various problems anise. First of all, it is known that yeasts themselveshave various sugar chain structures (K. Wolf et al., NonconventionalYeasts in Biotechnology (1995) ).

For example, a divided yeast Schizosaccharomyces pombe containsgalactose. Kluyveromyces lactis has GlcNAc. Both the methylotrophicyeast Pichia pastoris and the pathogenic yeast Candida albicans havebeen confirmed to contain β-mannoside linkage. Even yeasts having xyloseand rhamnose as sugar chain components exist (Biochim. et Biophy. Acta,1426, 1999, 227-237).

In fact, no yeasts capable of producing mammalian type sugar chains havebeen obtained except Saccharomyces cerevisiae as reported by Jigami etal. Also, although use of a methylotrophic yeast as the host forproducing a foreign protein was exemplified in Japanese PatentPublication (Kokai) No. 9-3097A (1997), substantially no other examplehas been given.

In Japanese Patent Publication (Kokai) No. 9-3097A (1997), a homologueof Pichia pastoris OCH1 gene and a Pichia pastoris mutant strain inwhich the OCH1 gene was knockout were prepared, to obtain from them amodified methylotrophic yeast strain whose ability to extend a sugarchain was inhibited as compared with natural methylotrophic yeaststrain. This publication, however, provides only information on SDS-PAGEof the produced glycoprotein, and no such support as structural analysisdata. That is, it did not actually identify the activity but onlypointed out about possibility of being α-1,6-mannosyl transferase. Infact, although HOC1 gene (GenBank accession number; U62942), which is anOCH1 gene homologue, exists also in Saccharomyces cerevisiae, theactivity and function thereof are unknown at present.

Moreover, in the same publication a sugar chain having β-mannosidelinkage in P. pastoris was identified, but it did not describe about thestructure of the chain in any way. Indeed, structural analysis of thesugar chain was neither performed nor identified the produced sugarchain. So, it was not demonstrated whether or not the obtained gene isactually the OCH1 gene, and whether or not the sugar chain of theknockout strain was a mammalian type. Accordingly, one cannot safely saythat the technique disclosed in Japanese Patent Publication (Kokai) No.9-3097A (1997) produces a mammalian type sugar chain bearingglycoprotein and is sufficient as the production system that can beadapted for production of medicaments.

There is also a study using a filamentous fungus Trichoderma reesei byMaras et al. as an attempt to produce a mammalian type sugar chain usinga microorganism other than yeast (U.S. Pat. No. 5,834,251). Thedisclosed method comprises making α-1,2-mannosidase and GnT-I to act onfilamentous fungus and yeast to synthesize a hybrid type sugar chain(i.e., GN1Man5 sugar chain).

Filamentous fungi inherently express α-1,2-mannosidase, and consequentlyit is believed that little sugar chain modification occurs as comparedwith the case of yeast. On the other hand, since yeast attaches aparticular outer sugar chain, all sugar chains are not obtained as Man5by the procedure in which only α-1,2-mannosidase is introduced. In fact,produced in Saccharomyces cerevisiae as disclosed in this patentpublication was a mixture of Man5 as the final product with sugar chainsof Man6 or more as partial decomposition products, which mixture isproduce by action of the outer sugar chain synthesizing gene OCH1, asdescribed by Jigami or Chiba et al. (supra). It would accordingly behard to say that the mammalian type sugar chain was produced in S.cerevisiae, and so this purpose cannot be attained without disrupting asugar chain biosynthesizing gene of yeast. Maras et al. did not mentionthe gene disruption in the sugar chain biosynthesis system inherent toyeast at all, so obviously this technique could not be applied to yeasts(Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis,Saccharomyces cerevisiae, Yarrowia lipolytica). Moreover, Maras et al.refers to RNaseB as a heterologous expression protein in the Examples,but RNaseB has originally a high mannose type sugar chain of Man5 orMan6. Many of the sugar chains of the animal cell origin are complextype sugar chains having complicated structures, and many ofglycoproteins such as cytokines expected to be applied to medicamentsetc. have complex type sugar chains. In fact, it is known that the sugarchain structure changes greatly depending on kinds of foreignglycoproteins expressed (Method in Molecular Biologylogy, 103, 95-105(1998)). Therefore, it is considered inappropriate to use as an exampleRNaseB which is a glycoprotein originally having a high mannose typesugar chain, in the application to glycoproteins having complex typesugar chains.

Furthermore, filamentous fungi are commonly used for the production ofindustrial enzymes, food enzymes, etc., and the transformation system isestablished, and production of enzymes by DNA recombinant technology hasalso been conducted. Nevertheless, there are the followingdisadvantages:

-   1) Since the protease activity is very strong, proteins produced are    prone to receive limited proteolysis.-   2) Since the fungi produce many proteins secreted outside the cell,    they are unsuitable for the production of proteinous medicaments    where homogeneity would be required.

Ogataea minuta as defined in the present invention is a strain oncereferred to as Pichia minuta or Hansenulla minuta, and was named Ogataeaminuta by Ogata et al. (Biosci. Biotecnol. Biochem., 58, 1245-1257(1994)). Ogataea minuta produces significant amounts of alcohol oxidase,dihydroxyacetone synthase and the formate dehydrogenase within the cellas in other methylotrophic yeasts, but nothing was known about the genesrelating to these methanol utilization enzyme nor about sugar chainstructures of this yeast.

Under the above-mentioned circumstances, the object of the presentinvention is to solve the above-described problems in production ofglycoproteins in yeast, and to provide a process for mass production ofnon-antigenic mammalian type sugar chains and glycoproteins containingthe sugar chains using a methylotrophic yeast wherein the sugar chainstructures are identical to those of sugar chains as produced in humanand other mammalian cells.

DISCLOSURE OF THE INVENTION

For the purpose of constructing a production technique of glycoproteinshaving mammalian cell compatible sugar chain structures using amethylotrophic yeast, we conducted intensive researches to achieve theabove-mentioned object. Consequently, we have found that sugar chains inOgataea minuta, which is a kind of methylotrophic yeast, comprisesmainly α-1,2-mannoside linkage, by NMR analysis of the cell wall sugarchain and by α-1,2-mannosidase digestion test, and further thatglycoproteins having mammalian type sugar chains can be obtained byintroducing an α-1,2-mannosidase gene into a mutant strain comprisingmutated sugar chain biosynthesizing enzyme genes (for example, an OCH1gene (α-1,6-mannosyl transferase) knockout mutant, which is consideredto be a key enzyme for the elongation reaction where mannose residuesattach to an M8 high mannose type sugar chain one by one via α-1,6linkage), and expressing it under the control of a potent promoter suchas methanol-inducible promoter, followed by culturing the Ogataea minutatransformed with a heterologous gene in a culture medium, thereby toobtain a glycoprotein from the culture. By this finding was completedthe present invention. Thus, it was found that a mammalian type sugarchain could be produced without disrupting MNN1 and MNN4 genes inSaccharomyces cerevisiae.

In summary, the invention comprises:

-   1) A methylotrophic yeast strain producing a mammalian type sugar    chain, obtained by introducing an α-1,2-mannosidase gene into a    mutant strain comprising a mutated sugar chain biosynthesizing    enzyme gene (for example, an OCH1 gene (α-1,6-mannosyl transferase)    knockout mutant, which is considered to be a key enzyme for the    elongation reaction where mannose residues attach to an M8 high    mannose type sugar chain one by one via α-1,6 linkage), and    expressing it under the control of a potent promoter such as    methanol-inducible promoter;-   2) A process of producing a glycoprotein comprising a mammalian type    sugar chain, comprising culturing in a culture medium the yeast    strain bred by introducing heterologous genes into a mutant yeast    which comprises mutated sugar chain biosynthesizing enzyme genes and    expressing these genes, and obtaining the glycoprotein comprising a    mammalian sugar chain from the culture; and-   3) A glycoprotein comprising a mammalian type sugar chain, produced    by this production process.

More specifically, the invention provides the following 1 to 122.

-   1. A process for producing a methylotrophic yeast capable of    producing a mammalian type sugar chain, which comprises the steps of-   1) disrupting an OCH1 gene which encodes α-1,6-mannosyl transferase,    in a methylotrophic yeast; and-   2) introducing an α-1,2-mannosidase gene into the yeast and    expressing it therein.-   2. A process according to (1), wherein the mammalian type sugar    chain is represented by the following structural formula    (Man₅GlcNAc₂):

-   3. A process according to (1) or (2), wherein the methylotrophic    yeast belongs to the genus Pichia, Hansenula, Candida, or Ogataea.-   4. A process according to (1) or (2), wherein the methylotrophic    yeast is Ogataea minuta.-   5. A process according to any one of (1) to (4), wherein the    methylotrophic yeast is a strain from Ogataea minuta strain IFO    10746.-   6. A process according to any one of (1) to (5), wherein the    α-1,2-mannosidase gene is expressed under the control of a    methanol-inducible promoter.-   7. A process according to (6), wherein the methanol-inducible    promoter is a promoter of an alcohol oxidase (AOX) gene.-   8. A process according to (7), wherein the alcohol oxidase (AOA)    gene is from Ogataea minuta.-   9. A process according to any one of (1) to (8), characterized in    that the α-1,2-mannosidase gene to be introduced is attached to a    yeast endoplasmic reticulum (ER) retention signal (HDEL) (SEQ ID NO:    121).-   10. A process according to any one of (1) to (9), wherein the    α-1,2-mannosidase gene is from Aspergillus saitoi.-   11. A process according to any one of (1) to (10), which further    comprises a step of transforming a heterologous gene into the yeast.-   12. A process according to (11), wherein the heterologous gene is    transferred using an expression vector and is expressed in the    yeast.-   13. A process according to (12), wherein the expression vector    comprises a methanol-inducible promoter.-   14. A process according to (13), wherein the methanol-inducible    promoter is a promoter of an alcohol oxidase (AOX) gene.-   15. A process according to (14), wherein the alcohol oxidase (AOX)    gene is from Ogataea minuta.-   16. A process according to (12), wherein the expression vector    comprises a promoter of a glyceraldehyde-3-phosphate dehydrogenase    (GAPDH) gene.-   17. A process according to any one of (11) to (16), wherein 20% or    more of N-linked sugar chains produced of the protein encoded by a    heterologous gene is the mammalian type sugar chain represented by    Structural Formula 2.-   18. A process according to any one of (11) to (16), wherein 40% or    more of N-linked sugar chains produced of the protein encoded by a    heterologous gene is the mammalian type sugar chain represented by    Structural Formula 2.-   19. A process according to any one of (11) to (16), wherein 60% or    more of N-linked sugar chains produced of the protein encoded by a    heterologous gene is the mammalian type sugar chain represented by    Structural Formula 2.-   20. A process according to any one of (11) to (16), wherein 80% or    more of N-linked sugar chains produced of the protein encoded by a    heterologous gene is the mammalian type sugar chain represented by    Structural Formula 2.-   21. A process according to any one of (11) to (20), wherein the    protein encoded by a heterologous gene is from humans.-   22. A process according to any one of (11) to (21), wherein the    protein encoded by a heterologous gene is an antibody or a fragment    thereof.-   23. A methylotrophic yeast produced by a process according to any    one of (1) to (22).-   24. A process for producing a protein encoded by a heterologous    gene, wherein the process comprises culturing the methylotrophic    yeast of (23) in a medium to obtain the protein encoded by a    heterologous gene comprising a mammalian type sugar chain from the    culture.-   25. A protein comprising a mammalian type sugar chain encoded by a    heterologous gene, wherein the protein is produced by the process of    (24).-   26. An orotidine-5′-phosphate decarboxylase (URA3) gene DNA encoding    an amino acid sequence substantially represented by SEQ ID NO: 16.-   27. A URA3 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO: 15.-   28. A recombinant expression vector substantially comprising the    gene DNA of (26) or (27) or a fragment thereof as a selectable    marker.-   29. An Ogataea minuta strain transformed with a recombinant    expression vector of (28).-   30. An Ogataea minuta strain according to (29), the strain being    from the strain IFO 10746.-   31. A phosphoribosyl-amino-imidazole succinocarboxamide synthase    (ADE1) gene DNA encoding an amino acid sequence substantially    represented by SEQ ID NO:28.-   32. An ADE1 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:27.-   33. A recombinant expression vector substantially comprising the    gene DNA of (31) or (32) or a fragment thereof as a selectable    marker.-   34. An Ogataea minuta strain transformed with the recombinant    expression vector of (33).-   35. An Ogataea minuta strain according to (34), the strain being    from the strain IFO 10746.-   36. An imidazole-glycerol-phosphate dehydratase (HIS3) gene DNA    encoding an amino acid sequence substantially represented by SEQ ID    NO:100.-   37. An HIS3 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:99.-   38. A recombinant expression vector substantially comprising the    gene DNA of (36) or (37) or a fragment thereof as a selectable    marker.-   39. A Ogataea minuta strain transformed with a recombinant    expression vector of (38).-   40. An Ogataea minuta train according to (39), the strain being from    the strain IFO 10746.-   41. A 3-isopropylmalate dehydrogenase (LEU2) gene DNA encoding an    amino acid sequence substantially represented by SEQ ID NO:108.-   42. A LEU2 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:107.-   43. A recombinant expression vector substantially comprising the    gene DNA of (41) or (42) or a fragment thereof as a selectable    marker.-   44. An Ogataea minuta strain transformed with the recombinant    expression vector of (43).-   45. An Ogataea minuta stain according to claim 44, the strain being    from the IFO 10746.-   46. An α-1,6-mannosyl transferase (OCH1) gene DNA encoding an amino    acid sequence substantially represented by SEQ ID NO:43.-   47. An OCH1 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:42.-   48. An Ogataea minuta strain wherein the gene of (46) or (47) has    been disrupted.-   49. An Ogataea minuta strain according to (48), the strain being    from the strain IFO 10746 strain.-   50. A proteinase A (PEP4) gene DNA encoding an amino acid sequence    substantially represented by SEQ ID NO:52.-   51. A PEP4 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:51.-   52. An Ogataea minuta strain wherein the gene of (50) or (51) has    been disrupted.-   53. An Ogataea minuta strain according to (52), the strain being    from the strain IFO 10746.-   54. A proteinase B (PRB1) gene DNA encoding an amino acid sequence    substantially represented by SEQ ID NO:58.-   55. A PRB1 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:57.-   56. An Ogataea minuta strain wherein the gene of (54) or (55) has    been disrupted.-   57. An Ogataea minuta strain according to (56), the strain being    from the strain IFO 10746.-   58. A YPS1 gene DNA encoding an amino acid sequence substantially    represented by SEQ ID NO:116.-   59. A YPS1 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:115.-   60. An Ogataea minuta strain wherein the gene of (58) or (59) has    been disrupted.-   61. An Ogataea minuta strain according to (60), the strain being    from the strain IFO 10746.-   62. A process for producing a protein encoded by a heterologous    gene, wherein the heterologous gene is transferred into the Ogataea    minuta strain of (60) or (61).-   63. A process according to (62), wherein the heterologous gene    encodes an antibody or a fragment thereof.-   64. A process for preventing from decomposition of an antibody or a    fragment thereof, comprising disrupting a YPS1 gene in a    methylotrophic yeast.-   65. A process according to (64), wherein the methylotrophic yeast is    an Ogataea minuta strain.-   66. A process according to (65), wherein the Ogataea minuta strain    is from the strain IFO 10746.-   67. A process according to any one of (64) to (66), wherein the    class of the antibody is IgG.-   68. A process according to (67), wherein the subclass of the    antibody is IgG1.-   69. A process according to any one of (64) to (68), wherein the    antibody is a human antibody.-   70. A KTR1 gene DNA encoding an amino acid sequence substantially    represented by SEQ ID NO:64.-   71. A KTR1 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:63.-   72. An Ogataea minuta strain wherein the gene of (70) or (71) has    been disrupted.-   73. An Ogataea minuta strain according to (72), the strain being    from the strain IFO 10746.-   74. An MNN9 gene DNA encoding an amino acid sequence substantially    represented by SEQ ID NO:70.-   75. An MNN9 gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:69.-   76. An Ogataea minuta strain wherein the gene of (74) or (75) has    been disrupted.

77. An Ogataea minuta strain according to claim 76, the strain beingfrom the strain IFO 10746.

-   78. An alcohol oxidase (AOX) gene DNA encoding an amino acid    sequence substantially represented by SEQ ID NO:78.-   79. An AOX gene DNA comprising a nucleotide sequence substantially    represented by SEQ ID NO:77.-   80. A DNA comprising a promoter of alcohol oxidase (AOX), wherein    the DNA comprises a nucleotide sequence substantially represented by    SEQ ID NO:79.-   81. A DNA comprising a terminator of alcohol oxidase (AOX), wherein    the DNA comprises a nucleotide sequence substantially represented by    SEQ ID NO:80.-   82. A gene expression cassette comprising a DNA comprising a    promoter as defined in (80), a heterologous gene, and a DNA    comprising a terminator as defined in (81).-   83. A recombinant expression vector comprising a gene expression    cassette of (82).-   84. An Ogataea minuta strain transformed with the recombinant    expression vector of (83).-   85. An Ogataea minuta strain according to (84), the strain being    from the strain IFO 10746.-   86. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene DNA    encoding an amino acid sequence substantially represented by SEQ ID    NO:6.-   87. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene DNA    comprising a nucleotide sequence substantially represented by SEQ ID    NO:5.-   88. A DNA comprising a promoter of glyceraldehyde-3-phosphate    dehydrogenase (GAPDH), wherein the DNA comprises an amino acid    sequence substantially represented by SEQ ID NO:7.-   89. A DNA comprising a terminator of glyceraldehyde-3-phosphate    dehydrogenase (GAPDH), wherein the DNA comprises an amino acid    sequence substantially represented by SEQ ID NO:8.-   90. A gene expression cassette comprising a DNA comprising a    promoter as defined in (88), a heterologous gene, and a DNA    comprising a terminator as defined in (89).-   91. A recombinant expression vector comprising the gene expression    cassette of (90).-   92. An Ogataea minuta strain transformed with a recombinant    expression vector of (91).-   93. An Ogataea minuta strain according to claim 92, the strain being    from the strain IFO 10746.-   94. A process for producing an Ogataea minuta strain, which is    capable of producing a mammalian type sugar chain represented by the    following structural formula (Man₅GlcNAc₂):

comprising a step of disrupting OCH1 gene (SEQ ID NO:42) in the Ogataeaminuta strain.

-   95. A process of (94), wherein the Ogataea minuta strain is from the    strain IFO 10746.-   96. A process according to (94) or (95), which further comprises a    step of disrupting at least one gene selected from the group    consisting of a URA3 gene comprising the nucleotide sequence    represented by SEQ ID NO:15, an ADE1 gene comprising the nucleotide    sequence represented by SEQ ID NO:27, an HIS3 gene comprising the    nucleotide sequence represented by SEQ ID NO:99, and a LEU2 gene    comprising the nucleotide sequence represented by SEQ ID NO:107.-   97. A process according to any one of (94) to (96), which further    comprises a step of disrupting at least one gene selected from the    group consisting of a PEP4 gene comprising the nucleotide sequence    represented by SEQ ID NO:51, a PRB1 gene comprising the nucleotide    sequence represented by SEQ ID NO:57, and a YPS1 gene comprising the    nucleotide sequence represented by SEQ ID NO:115.-   98. A process according to any one of (94) to (97), which further    comprises a step of disrupting a KTR1 gene comprising the nucleotide    sequence represented by SEQ ID NO:63 and/or an MNN9 gene comprising    the sequence represented by SEQ ID NO:69.-   99. A process according to any one of (94) to (98), which further    comprises a step of introducing and expressing an α-1,2-mannosidase    gene from Aspergillus saitoi.-   100. A process according to (99), wherein the α-1,2-mannosidase gene    is transferred into the vector of (83) and expressed.-   101. A process according to any one of (94) to (100), which further    comprises a step of introducing and expressing a PDI gene.-   102. A process according to (101), wherein the PDI gene is a gene    (M62815) from Saccharomyces cerevisiae.-   103. A process according to (101) or (102), wherein the PDI gene is    transferred into the vector of claim 83 and expressed.-   104. A process according to any one of (94) to (103), which further    comprises a step of introducing and expressing a heterologous gene.-   105. A process according to (104), wherein the heterologous gene is    transferred into the vector of claim 83 and expressed.-   106. A process for producing a protein encoded by a heterologous    gene, which comprises culturing Ogataea minuta produced by the    process of (104) or (105) in a medium, to obtain the protein    comprising a mammalian type sugar chain encoded by the heterologous    gene from the culture.-   107. A protein comprising a mammalian type sugar chain encoded by a    heterologous gene, wherein the protein has been produced by the    process of (106).-   108. A process for producing an Ogataea minuta strain, which is    capable of producing a mammalian type sugar chain represented by the    following structural formula (Man₅GlcNAc₂):

wherein the process comprises the steps of:

-   -   disrupting an OCH1 gene represented by SEQ ID NO:42 in an        Ogataea minuta strain; and    -   disrupting a URA3 gene represented by SEQ ID NO:15 in the same        strain; and    -   disrupting a PEP4 gene represented by SEQ ID NO:51 in the same        strain; and    -   disrupting a PRB1 gene represented by SEQ ID NO:57 in the same        strain.

-   109. A process according to (108), wherein the Ogataea minuta strain    is from the strain IFO 10746.

-   110. A process according to (108) or (109), which further comprises    a step of disrupting an ADE1 gene comprising the nucleotide sequence    represented by SEQ ID NO:27.

-   111. A process according to (110), which further comprises a step of    disrupting a KTR1 gene comprising the nucleotide sequence    represented by SEQ ID NO:63.

-   112. A process according to (111), which further comprises a step of    disrupting an HIS3 gene comprising the nucleotide sequence    represented by SEQ ID NO:99.

-   113. A process according to (111), which further comprises a step of    disrupting a LEU2 gene comprising the nucleotide sequence    represented by SEQ ID NO:107.

-   114. A process according to (111), which further comprises the step    of:

-   1) disrupting a YPS1 gene comprising the nucleotide sequence    represented by SEQ ID NO:115.

-   115. A process according to any one of (108) to (114), which further    comprises a step of introducing and expressing an α-1,2-mannosidase    gene.

-   116. A process according to (115), wherein the α-1,2-mannosidase    gene is transferred into the vector of (83) and expressed.

-   117. A process according to any one of claims 108 to 116, which    further comprises a step of introducing and expressing a PDI gene    (M62815).

-   118. A process according to (117), wherein the PDI gene (M62815) is    transferred into the vector of (83) and expressed.

-   119. A process according to any one of (108) to (118), which further    comprises a step of introducing and expressing a heterologous gene.

-   120. A process according to claim 119, wherein the heterologous gene    is transferred into the vector of (83) and expressed.

-   121. A process for producing a protein encoded by a heterologous    gene comprising a mammalian type sugar chain, wherein the process    comprises culturing Ogataea minuta produced by the process of (119)    or (120) in a medium to obtain the protein from the culture.

-   122. A protein encoded by a heterologous gene comprising a mammalian    type sugar chain, wherein the protein has been produced by the    process of(121).

This specification includes the contents disclosed by the specificationand/or drawings of the Japanese Patent Application No. 2002- 127677,which is the basis of the priority claim of this application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the biosynthesis pathway of N-linked sugar chains, which isgeneral in mammals.

FIG. 2 shows the biosynthesis pathway of N-linked sugar chains in yeast(S. cerevisiae), wherein M is mannose, and α2, α3, α6 and β4 mean α-1,2linkage, α-1,3 linkage, α-1,6 linkage and β-1,4 linkage, respectively.

FIG. 3 shows the ¹H-NMR analysis of cell wall sugar chains of variousyeasts.

FIG. 4 shows the HPLC (amide column) analysis of digests which wereobtained by digesting sugar chains prepared from mannoproteins of cellwalls of various yeasts by Aspergillus saitoi α-1,2-mannosidase (productof Seikagaku Corporation).

FIG. 5 shows the restriction maps of plasmids pOMGP1, pOMGP2, pOMGP3 andpOMGP4.

FIG. 6 shows the restriction maps of plasmids pOMUR1, pOMUM1 and pDOMU1.

FIG. 7 shows the structures of the URA3 loci of a wild strain of Ogataeaminuta, a strain transformed with plasmid pDOMU1 and a URA3 geneknockout mutant, along with positions of PCR primers.

FIG. 8 shows the restriction maps of plasmids pOMAD1 and pDOMAD1. Therestriction enzyme sites added artificially are underlined.

FIG. 9 shows the restriction maps of plasmids pOMUR2 and pROMU1.

FIG. 10 shows the structures of the ADE1 loci of a wild strain ofOgataea minuta, an ADE1 gene knockout mutant disrupted by plasmidpDOMAD1, and a URA3 gene deficient mutant, along with positions of PCRprimers.

FIG. 11 shows the restriction maps of plasmids pOMOC1, pOMOC2B, pOMOC3Hand pDOMOCH1. The restriction enzyme sites of the vector are underlined.

FIG. 12 shows the structures of the OCH1 gene loci of a wild strain ofOgataea minuta, an OCH1 gene knockout mutant disrupted by the plasmidpDOMOCH1, and a URA3 gene deficient mutant, along with positions of PCRprimers.

FIG. 13 shows the structure analysis by an amide and reverse phasecolumns for sugar chains of the mannan glycoproteins of Ogataea minutastrain TK3-A which is an OCH1 gene knockout mutant and of its parentstrain Ogataea minuta strain TK1-3.

FIG. 14 shows the restriction maps of plasmids pOMPA1 and pDOMPA1, andthe structures of the PEP4 loci of a wild strain of Ogataea minuta, aPEP4 gene knockout mutant disrupted by plasmid pDOMPA1, and a URA3 genedeficient mutant. The restriction enzyme sites of the vector origin areunderlined.

FIG. 15 shows the restriction maps of plasmids pOMPB1 and pDOMPB1, andthe structures of the PRB1 loci of a wild strain of Ogataea minuta, aPRB1 gene knockout mutant disrupted by plasmid pDOMPB1, and a URA3 genedeficient mutant.

FIG. 16 the restriction maps of plasmids pOMKR1 and pDOMKR1, and thestructures of the KTR1 loci of a wild strain of Ogataea minuta, a KTR1gene knockout mutant disrupted by plasmid pDOMKR1, and a URA3 genedeficient mutant. The restriction enzyme sites of the vector areunderlined.

FIG. 17 shows the restriction maps of plasmids pOMMN9-1 and pDOMN9, andthe structures of the MNN9 loci of a wild strain of Ogataea minuta, anMNN9 gene knockout mutant disrupted by the plasmid pDOMN9 and a URA3gene deficient mutant, along with positions of PCR primers.

FIGS. 18A and 18B show the restriction maps of plasmids pOMAX1,pOMAXPT1, pOMUR5, pOMUR6, pOMUR-X, pOMUR-XN, pOMex1U, pOMex2U, pOMex3G,pOMex4A, pOMex5H, pOMexGP1U and pOMexGP4A. The restriction enzyme sitesof the vector are underlined.

FIG. 19 shows the structure analysis by amide and reverse phase columnsfor sugar chains of the mannan glycoprotein of Ogataea minuta strainTK3-A-MU1, which is an och1Δ strain expressing an Aspergillussaitoi-derived α-1,2-mannosidase gene.

FIG. 20 shows the structure analysis by amide and reverse phase columnsof the Saccharomyces cerevisiae-derived invertase produced by Ogataeaminuta strain TK3-A-MU-IVG1, which is an Ogataea minuta OCH1 geneknockout mutant expressing Aspergillus saitoi-derived α-1,2-mannosidasegene.

FIG. 21 shows the Western analysis of the antibody produced by usingOgataea minuta strain TK9-IgB-aM.

FIG. 22 shows the purification of the antibody produced by using Ogataeaminuta strain TK9-IgB-aM.

FIG. 23 shows the binding activity to G-CSF of the antibody produced byusing Ogataea minuta strain TK9-IgB-aM.

FIG. 24 shows the analysis of the sugar chains of antibodies produced byusing Ogataea minuta strain TK9-IgB and Ogataea minuta strainTK9-IgB-aM.

FIG. 25 shows the restriction maps of plasmids pOMHI1, pOMHI2, pOMHI3,pOMHI4 and pDOMHI1. The restriction enzyme sites of the vector andlinker are underlined.

FIG. 26 shows the structures of the HIS3 loci of a wild strain ofOgataea minuta, an HIS3 gene knockout mutant disrupted by plasmidpDOMHI1, and a URA3 gene deficient mutant, along with positions of PCRprimers.

FIG. 27 shows the construction of plasmid pOMex6HS and its restrictionmap. The restriction enzyme sites of the vector and linker areunderlined.

FIG. 28 shows the restriction maps of plasmids pOMLE1, pOMLE2 andpDOMLE1. The restriction enzyme sites of the vector and linker areunderlined.

FIG. 29 shows the structures of the LEU2 loci of a wild strain ofOgataea minuta, a LEU2 gene knockout mutant disrupted by the plasmidpDOMLE1, and a URA3 gene deficient mutant, along with positions of PCRprimers.

FIG. 30 shows the construction of plasmid pOMex7L and its restrictionmap. The restriction enzyme sites of the vector and linker areunderlined.

FIG. 31 shows the restriction maps of plasmids pOMYP1, pOMYP2, pOMYP3and pDOMYP1. The restriction enzyme sites of the vector and linker areunderlined.

FIG. 32 shows the structures of the YPS1 loci of a wild strain ofOgataea minuta, a YPSI gene knockout mutant disrupted by plasmid pDOMLE1and a URA3 gene deficient mutant, along with positions of PCR primers.

FIG. 33 shows the Western analysis of the antibody produced by usingOgataea minuta strain YK3-IgB-aM.

FIG. 34 shows the purification of the antibody produced by using Ogataeaminuta strain YK3-IgB-aM (Western analysis, and reducing & non-reducingcondition).

FIG. 35 shows the Western analysis of the antibody produced by usingOgataea minuta strain YK3-IgB-aM-PDI.

ABBREVIATION

GlcNAc, GN: N-acetylglucosamine

Man, M: mannose

PA: 2-amino pyridylation

MODES FOR CARRYING OUT THE INVENTION

Hereinafter, the invention will be described in detail.

According to the invention, the process for producing a glycoproteincomprising a mammalian type sugar chain(s) comprises the following stepsof:

-   1) breeding a methylotrophic yeast strain producing a mammalian type    sugar chain, by introducing an α-1,2-mannosidase gene into a mutant    strain comprising mutated sugar chain biosynthesizing enzyme genes    (for example, an OCH1 gene α-1,6-mannosyl transferase) knockout    mutant, which is considered to be a key enzyme for the elongation    reaction where mannose residues attach to an M8 high mannose type    sugar chain one by one via α-1,6 linkage), and expressing it under    the control of a potent promoter such as methanol-inducible    promoter; and-   2) culturing in a medium the yeast strain bred by introducing    heterologous genes into a mutant yeast which comprises mutated sugar    chain biosynthesizing enzyme genes and expressing these genes, and    obtaining the glycoproteins comprising a mammalian sugar chain from    the culture.    1. Preparation of Mammalian Type Sugar Chain Producing Strains

According to the present invention, mutant strains of yeast capable ofproducing mammalian type sugar chains, wherein the mutant strain has adisruption in its outer chain biosynthesis gene specific to yeast andhas been deprived of sugar chains specific to yeast, can be prepared inthe following manner.

1-1 Preparation of Man5 Type Sugar Chain (“High Mannose Type SugarChain”) Producing Yeasts

Mutation trait necessary for the mutant yeast of the invention is amutation of a gene(s) peculiar to yeast associated with the outer sugarchain biosynthesis system, and specifically at least a mutation of OCH1gene. That is, as long as the mutant yeast has the above-mentionedmutation, it may be either a natural mutant strain or an artificialmutant strain.

The OCH1 gene means a gene encoding α-1,6 mannosyl transferase, whichcatalyses the initial reaction of the outer sugar chain formation inyeast, and works to further transfer a mannose residue to the core sugarchain of N-linked sugar chain of a glycoprotein of yeast viaα-1,6-linkage. This reaction functions as a trigger for attachingmannose excessively compared with the glycoproteins of animal cells(“hyper-mannosylation”), thereby forming a mannan-type sugar chainpeculiar to yeast. Therefore, OCH1 gene encodes a protein having theabove-mentioned activity and function strictly, and it does not refer toa gene which simply has a homology to the gene sequence or the aminoacid sequence deduced from the gene sequence.

However, in order to change the sugar chain of yeast into a mammaliantype sugar chain, just the manipulation that disrupts this OCH1 gene isnot enough.

As mentioned above, in a mammalian cell, α-mannosidase I acts on a highmannose type sugar chain to cut off several mannose residues, andfinally generates a Man5 high mannose type sugar chain (“Man5GlcNAc2”).This Man5 type sugar chain serves as a prototype of mammalian type sugarchain. N-acetylglucosaminyl transferase (GnT) I acts on this sugarchain, and causes the transfer of one N-acetylglucosamine residue togenerate a hybrid type sugar chain which comprises GlcNAcMan5GlcNAc2,followed by successive formation of complex type sugar chains.Therefore, to make a yeast cell to produce a mammalian type sugarchain(s), it would be necessary to create a yeast which produces a Man5high mannose type sugar chain (i.e., Man5GlcNAc2) first.

α-1,2-mannosidase (also referred to as α-mannosidase-I) as used in theinvention is not limited as long as it has the above-mentioned enzymeactivity. For example, α-mannosidase-I involved in the above-mentionedsugar chain biosynthesis system in mammalian cells, α-mannosidaseenzymes from other animals such as nematode, and α-1,2-mannosidaseenzymes from fungi such as Aspergillus saitoi can be used.

In order to effect the invention efficiently, the expression site ofα-1,2-mannosidase is important. It is said that α-1,2-mannosidasefunctions in the cis Golgi in mammalian cells. On the other hand,addition of a sugar chain peculiar to yeast in the yeast cell isperformed in the cis, medial or trans Golgi. Therefore, it is necessaryto make α-1,2-mannosidase act prior to the modification in which a sugarchain peculiar to yeast is attached, i.e., modification in Golgiapparatus. If the expression site is in the Golgi apparatus which existsdownstream in the transportation pathway of glycoprotein, then Man5 typesugar chains cannot be generated efficiently.

Therefore, to attain this purpose, endoplasmic reticulum (ER) retentionsignal (for example, amino acid sequence shown by His-Asp-Glu-Leu) inyeast may be attached to the C terminus of the protein ofα-1,2-mannosidase thereby localizing the enzyme within ER to causeexpression of the activity so that the attachment of sugar chainpeculiar to yeast can be inhibited. This method was already reported byinventors (Chiba et al., J. Biol. Chem., 273, 26298-26304 (1998)).

However, when the sugar chain of a certain protein is changed into amammalian type sugar chain in order to use this protein as a drug, it isrequired to remove sugar chains peculiar to yeast almost completely, anduse of only the above-mentioned technique is supposed to beinsufficient. In fact, although in the above-mentioned report Chiba etal. use the promoter of glyceraldehyde-3-phosphate dehydrogenase, whichis known to be the strongest promoter functioning in Saccharomycescerevisiae, in the expression of the glyceraldehyde-3-phosphatedehydrogenase, the results of analyzing the sugar chains of cell wallglycoproteins reveal that Man5 type sugar chains were generated in thelevel of only about 10%.

The system using the sugar chain mutant of Ogataea minuta in theinvention enables formation of a Man5 type sugar chain in the amount of20% or more, preferably 40% or more, more preferably 60% or more, mostpreferably 80% or more of the sugar chains of the cell wallglycoproteins which the yeast produces as in the Examples below. Also,Man5 type sugar chains are formed in the amount of 20% or more,preferably 40% or more, more preferably 60% or more, most preferably 80%or more in the example of the secretion and expression of a heterologousgene. Thus the problems in Saccharomyces cerevisiae have been solved.The application of Ogataea minuta in the invention to variousglycoproteins will be expected from these results.

On the other hand, Chiba et al. uses the Δoch1Δmnn1Δmnn4 strain whichgenerates only the Man8 type sugar chain, a core sugar chain. MNN1 geneis presumed to be a gene peculiar to Saccharomyces cerevisiae, and thesugar chain synthesis pathway and sugar chain synthesizing genes wasisolated and analyzed, but sugar chain structure was not fully analyzedfor other yeasts. For example, the existence of a sugar chain which hasβ-mannoside linkage is known for Pichia pastoris as mentioned above(Higgins (ed.), Pichia Protocols, 1998, pp. 95-105, Humana Press andBiochim.et Biophy. Acta, 1426, 227-237 (1999)). Moreover, the results ofSDS-PAGE of the glycoproteins produced by the OCH1 gene homologueknockout mutant disclosed in Japanese Patent Publication (Kokai) No.9-3097A (1997) surely presented the data indicating that the sugarchains have been shortened into lower molecules; namely, it is presumedthat they are not glycoproteins having a single sugar chain like Man8type sugar chain. No gene involved in the synthesis of these sugarchains has been isolated, and great labors are needed for isolating anddisrupting the gene.

Thus, to allow a yeast strain to produce Man5 type sugar chains, it isnecessary to cause α-1,2-mannosidase to highly express, and for thispurpose, a potent promoter is needed. In these circumstances, theinvention was completed by using an alcohol oxidase (AOX) gene promoter(inducible by methanol) from methylotrophic yeast known as the strongestinducible expression promoter. Other inducible expression promotersusable in the invention include, but not limited to, promoters fordihydroxyacetone synthetase (DAS) gene and formate dehydrogenase (FDH)gene, and any promoter can used as long as it has an ability to expressthe enzyme gene in the methylotrophic yeast of the invention.

Thus, mammalian type sugar chains can be produced without disrupting anouter sugar chain synthesis gene peculiar to yeast, by preliminarilytrimming (removing) the sites on the sugar chain to which sugar chainspeculiar to yeast is attached in the ER and Golgi apparatus.Accordingly, the acquisition of a gene for forming β-mannoside linkageand of an MNN4 gene, which is for addition of mannose phosphate, becomesunnecessary.

However, OCH1 exists quite ubiquitously in yeast, and the locationthereof is relatively near the reducing terminal side of the core sugarchain and so it is believed that the gene should be destroyed in orderto remove its activity.

Yeast strains applicable to the invention include any strain in whichthe sugar chain of glycoprotein mainly comprises α-1,2-mannosidelinkage, and methylotrophic yeasts are not limited as long as theyproduce N-linked sugar chains which mainly comprise α-1,2-mannosidelinkage, including as specific examples Ogataea minuta, Candidasucciphila, Pichia pastoris, Pichia trehalophila, Pichia methanolica,Pichia angusta, Hansenulla polymorpha, etc. Preferred is Ogataea minuta.

Therefore, the procedures disclosed by the invention are inapplicable toyeast strains having the structure where sugar chains other than α-1,6mannose have been attached directly to the core sugar chain by the OCH1gene. That is, any yeast strain which generates glycoproteins with sugarchains peculiar to yeast attached to moieties of the core sugar chain,strictly to moieties of the Man5 type sugar chain, cannot utilize in theprocedures of the invention.

Furthermore, mammalization can be more efficiently attained by auxiliarydisruption of a KTR gene homologue belonging to (α-mannosyl transferasegene family (for example, KTR1 gene of Ogataea minuta as found in theinvention), or of an MNN9 gene homologue (for example, MNN9 gene ofOgataea minuta as found by the invention) which is believed to beinvolved in the attachment of sugar chains in the Golgi apparatus.

Furthermore, since sugar chain mutants have generally shorter sugarchains in glycoproteins, and as a result, the cell wall becomes weaker,so the drug susceptibility increases or the resistance to osmoticpressure decreases in the mutants. In such a case problems may occur incell culture. On the contrary, in the procedure of the invention, whichutilizes a methanol-inducible promoter and expresses α-1,2-mannosidase,mammalian type sugar chains can be produced as a by-product along with aglycoprotein encoded by a heterologous gene. Hence, the culture andproduction can be performed without applying a burden at the time ofmultiplication of the yeast cell.

The term “a gene(s) associated with the mammalian type sugar chainbiosynthesis” as described above means an appropriate number oftransgenes, which belong to a group of one or more of theabove-mentioned genes, required to produce a sugar chain of interest.When the transgenes are plural, they may belong to a group of homo-typegenes or to a group of hetero-type genes.

In order to obtain the produced sugar chains and glycoproteins in highyield, it is desirable to make the above-mentioned enzymes to expresshighly in a suitable organ (for example, Golgi apparatus). Therefore, itis effective to use genes compatible to the codon usage of yeast. Also,to localize the enzymes in a suitable organ, the addition of a signalsequence or the like of yeast will become effective. For the transfer ofa gene, use of vectors such as chromosome integration type (YIp type)vector may be considered. Promoters required to express the geneinclude, but are not limited to, constitutive expression promoters suchas GAPDH and PGK, inducible expression promoters such as AOX1, etc.However, since multiplication of yeast may be affected when one or moreglycosidase, glycosyltransferases, or sugar nucleotide transporter genesare expressed, it is necessary to take into consideration the use of aninducible promoter or the appropriate order of introducing genes.

The mutant yeast which produces the above-mentioned mammalian type sugarchain, or the mutant to which the above-mentioned foreign gene has beentransferred, is cultured in a culture medium, thereby to produceglycoproteins comprising the same Asn-linked sugar chain as thehigh-mannose type sugar chain (Man₅GlcNAc₂), the hybrid type sugar chain(GlcNAcMan₅GlcNAc₂) or the complex type sugar chain (for example,Gal₂GlcNAc₂Man₃GlcNAc₂), which the mammalian cell produces, eitherintracellularly or extracellularly. In this case, the content of anouter sugar chain peculiar to yeast is significantly reduced.

Specifically, the transfer of a GnT-I gene into the above-mentionedmutant enables production of a hybrid type sugar chain, and the transferof a gene(s) associated with the mammalian type sugar chain biosynthesissystem (α-mannosidase II, GnT-II, GalT, UDP-GlcNAc Transporter, and/orUDP-Gal Transporter genes) enables production of a double-strandedcomplex type sugar chain (Gal₂GlcNAc₂Man₂GlcNAc₂).

2. Different Genes from Ogataea minuta Usable in the Invention

The proteins usable in the invention are not particularly limited aslong as they have respective activities, and specifically they areproteins comprising an amino acid sequence substantially represented bythe SEQ ID NO described in the Examples below. As used herein, the term“an amino acid sequence substantially represented by SEQ ID NO:X” meansthat the amino acid sequence includes:

-   -   (a) the amino acid sequence represented by SEQ ID NO:X; or    -   (b) an amino acid sequence which comprises a deletion(s), a        substitution(s) or an addition(s) of one or several amino acids        in the amino acid sequence represented by SEQ ID NO:X.

That is, the above amino acid sequence may be partially modified (forexample, substitution, deletion, insertion or addition of an amino acidresidue(s) or a peptide chain(s), etc.). Herein, the term “several” inrelation to the number of deleted, substituted or added amino acidsmeans any number in the range capable of being introduced by the methodsusually used in art, preferably 2 to 10, more preferably 2 to 5, andmost preferably 2 to 3.

DNAs comprising a nucleotide sequence, which encodes the protein usablein the invention, are characterized by comprising the nucleotidesequences encoding the above-mentioned proteins from Ogataea minuta asdefined in the invention. Such nucleotide sequences are not particularlylimited as long as they are the nucleotide sequences encoding theproteins of the invention, and their examples are the nucleotidesequences which encode amino acid sequences substantially represented bythe SEQ ID NOs described in the Examples below. As used herein, the term“a nucleotide sequence substantially represented by SEQ ID NO:X” meansthat the nucleotide sequence includes:

-   -   (a) the nucleotide sequence represented by SEQ ID NO:X; or    -   (b) a nucleotide sequence comprising a deletion, a substitution        or an addition of one or several nucleotides in the nucleotide        sequence represented by SEQ ID NO:X.

This DNA may be conventionally produced by the known procedures. Forexample, all or part of the DNA may be synthesized by using a DNAsynthesizer based on the nucleotide sequence illustrated in theinvention, or may be prepared by PCR amplification using chromosome DNA.Here, the term “several” in relation to the number of deleted,substituted or added nucleotides means any number in the range capableof being introduced by the methods usually used in art, for example,site-directed mutagenesis (e.g., Molecular Cloning, A Laboratory Manual,second edition, ed. by Sambrook et al., Cold Spring Harbor LaboratoryPress, 1989; Current Protocols in Molecular Biology, John Wiley & Sons(1987-1997)), for example, 2 to 10, preferably 2 to 5, and morepreferably 2 to 3.

3. Obtaining Genes

Isolation of a target gene fragment can be performed by extractinggenomic DNA from a yeast strain, and selecting the target gene, by usinggeneral procedures (Molecular Cloning (1989), Methods in Enzymology 194(1991)). In the above, the genomic DNA from Ogataea minuta can beextracted, for example, by the methods of Cryer et al. (Methods in CellBiology, 12, 39-44 (1975)) and of P. Philippsen et al. (MethodsEnzymol., 194, 169-182 (1991)).

For example, the protoplast prepared from yeast can be subjected to aconventional DNA extraction method, an alcohol precipitation methodafter removing cell debris under high salt concentration, an alcoholprecipitation method after extracting with phenol and/or chloroform,etc. Besides the above method utilizing the preparation of protoplast,DNA may be extracted by break of cells with glass beads. The protoplastmethod is preferable because preparation of high molecular weight DNA iseasy.

A target gene can be obtained, for example, by the PCR method (PCRTechnology, Henry A. Erlich, Atockton press (1989)). The PCR is atechnique which enables in vitro amplification of a specific DNAfragment to hundreds of thousands fold or more in about 2 to 3 hours,using a combination of sense/antisense primers annealed at each end ofthe target region, a heat-resistant DNA polymerase, and a DNAamplification system. In the amplification of a target gene, 25-30mersynthetic single-stranded DNAs and genomic DNA can be used as primersand as a template, respectively. The amplified gene may be identified interms of its nucleotide sequence before use.

The DNA sequence of a gene can be determined by usual methods such as,for example, dideoxy method (Sanger et al., Proc. Natl. Acad. Sci., USA,74, 5463-5467 (1977)). Alternatively, the nucleotide sequence of DNA caneasily be determined by use of commercially available sequencing kits orthe like.

The isolation, purification, etc. of the DNA can also be carried out byordinary methods, and in the case of E. coli for example, the DNA may beextracted by the alkali/SDS method and ethanol precipitation, and theDNA subsequently purified by RNase treatment, PEG precipitation or thelike.

A target gene can also be obtained by: (a) extracting the total DNA ofthe above-mentioned yeast, transferring a gene transfer vector, whichcomprises a DNA fragment derived from said DNA, into a host, thereby toprepare a gene library of the yeast, and (b) subsequently selecting thedesired clone from the gene library, followed by amplifying the clone.

The gene library can be prepared as a genomic library by partiallydigesting the chromosomal DNA obtained by the above-mentioned methodwith appropriate restriction enzymes (such as Sau3AI) to obtainfragments thereof, ligating the fragments with an appropriate vector,and transforming an appropriate host with the vector. Alternatively, itis also possible by amplifying a fragment of the target gene by PCRfirst, screening for restriction sites by the genomic Southern analysisso that the target gene can be obtained efficiently, and digesting thechromosomal DNA by this restriction enzyme to obtain the desiredfragment. Vectors usable for this purpose include commercially availableplasmids such as pBR system, pUC system, Bluescript system, etc.,usually known as the known vectors for preparing a gene library. Phagevectors of Charon system or EMBL system etc. or cosmids can be also usedwidely. The host to be transformed or transduced with the preparedvector for preparation of gene library can be selected depending on thetype of the above-mentioned vectors.

Clones can be selected and obtained from the above-mentioned genelibrary using a labeled probe which comprises a sequence peculiar to atarget gene, by means of colony hybridization, plaque hybridization orthe like. A sequence peculiar to target gene used as a probe can beobtained by synthesizing a corresponding oligonucleotide of the genewhich encodes the amino acid sequence of a target protein purified fromOgataea minuta, specifically amplifying the desired DNA fragment by PCRusing the chromosomal DNA of Ogataea minuta as a template, to obtain it.The peculiar sequence may also be obtained by searching for a gene whichencodes a protein homolog from different species in DNA databases suchas GenBank or protein databases such as SWISS-PROT, to obtain thesequence information, synthesizing an oligonucleotide corresponding tothe conserved amino acid sequence analyzed with an analyzing softwaresuch as homology search programs such as BLAST, GENETYX (SoftwareDevelopment), and DNAsis (Hitachi Software), and specifically amplifyingthe desired DNA fragment by PCR using the chromosomal DNA of Ogataeaminuta as a template. The synthesized oligonucleotide may be used as aprobe. Once the nucleotide sequence is determined, the desired gene canbe obtained by chemical synthesis or PCR using primers synthesized basedon the determined nucleotide sequence, or by hybridization using as aprobe the DNA fragment comprising the above-mentioned nucleotidesequence.

4. Gene Disruption

In the invention, a target gene is basically disrupted in accordancewith the method disclosed by Rothstein, in Methods Enzymol., 101,202-211 (1983). Specifically, a target gene DNA obtained by theabove-described method is first cut or partially deleted, an appropriateselectable marker gene DNA is inserted at the cut or deleted site,thereby to prepare a DNA structure in which the selectable marker hasbeen sandwiched between upstream and downstream regions of the targetgene. Subsequently, this structure is transferred to a yeast cell. Theabove manipulation results in two recombinations at homologous moietiesbetween each end of the transferred fragment (i.e., the DNA structurewith a selectable marker sandwiched) and a target gene on chromosome,thereby substituting the target gene on chromosome with the transferredfragment. Auxotrophic markers and drug resistant markers, as shownbelow, may be used as the selectable marker for gene disruption. In thiscase, one selectable marker will generally be required for disruptingone gene. When URA43 gene is used, ura3 trait can be efficientlyreproduced and so it is often used for this purpose.

Specific explanation is provided using an example of the preparation ofan OCH1 gene knockout strain. A plasmid carrying URA3 gene, whichcomprises a repeated structure before and after structural gene, isconstructed, and the gene cassette cleaved out with a restriction enzymeis inserted at a target gene on the plasmid, thereby to construct adisrupted allele. Gene-knockout strain can be obtained by substitutingwith a target gene on the chromosome using this plasmid. As the URA3gene inserted into the chromosome is sandwiched by the repeatedstructures, it is dropped out of the chromosome due to homologousrecombination between the repeated structures. The selection of thisURA3 deficient strain can be carried out by use of 5-fluoroorotic acid(5-FOA). A ura3 mutant is resistant to 5-FOA (Boeke et al., Mol. Gen.Genet., 197, 345-346 (1984); Boeke et al., Methods Enzymol., 154,165-174 (1987)), and a cell strain having URA3+ phenotype can no longergrow in the 5-FOA medium. Thus, separating a strain with resistant traitin a medium to which 5-FOA is supplemented, enables manipulations usinga URA3 gene marker again. Therefore, the mutated auxotrophic trait ofthe original yeast strain is not damaged by gene destruction in the“artificial knockout mutant” which has undergone the gene disruptionartificially by this technique.

In addition, in the “natural mutant” where the gene disruption occursnaturally without using the above-mentioned procedures butspontaneously, the number of the mutated auxotrophic traits is notdecreased nor increased.

5. Marker for Gene Transfer

The auxotrophic marker for transfer of a heterologous gene into themutant yeast of the invention is defined by yeast strains to be used,and is specifically selected from ura3, his3, leu2, ade1 and trp1mutations. Although the number of auxotrophic markers depends on thenumber of transfer genes, generally one auxotrophic marker is requiredfor transfer of one gene. When plural of genes are transferred, a largernumber of auxotrophic markers become necessary as the number of transfergenes increases more and more, since the transfer gene fragment islonger, and transfer efficiency decreases, and as a result, expressionefficiency also decreases.

In the invention, the gene which complements the auxotrophy is a geneassociated with the in vivo synthetic system of biological componentssuch as amino acids and nucleic acids. The complementing gene is anoriginal functional gene itself, since the mutated traits include such amutation that the gene fails to function. Therefore, the gene from theoriginal yeast strain is desirable.

Usable selectable markers other than the above-mentioned auxotrophicmarkers include drug resistance markers, which impart resistance todrugs such as G418, cerulenin, aureobasidin, zeocin, canavanine,cycloheximide, hygromycin and blastcidin, and may be used to transferand disrupt a gene. Also, it is possible to perform the transfer anddisruption of a gene by using, as a marker, the gene which imparts asolvent resistance like ethanol resistance, an osmotic pressureresistance like resistance to salt or glycerol, and a metal ionresistance like resistance to copper, etc.

6. Method for Transfer of DNA into Cell and Transformation with Same

Methods for transferring a DNA into a cell for its transformation withthe DNA in the above procedures include general methods, for example, amethod of incorporating a plasmid into a cell after the cell is treatedwith lithium salt so that the DNA is prone to be naturally transferredinto the cell (Ito et al., Agric. Biol. Chem., 48, 341 (1984)), or amethod of electrically transferring a DNA into a cell, a protoplastmethod (Creggh et al., Mol. Cell. Biol., 5, 3376 (1985)), and the like(Becker and Guarente, Methods Enzymol., 194, 182-187 (1991)). Theexpression vector of the invention can be incorporated into the hostchromosome DNA, and can exist stably.

7. Expression of Heterologous Gene

The term “heterologous gene” as used herein is a gene of interest to beexpressed, and means any gene different from the gene for Ogataeaminuta-derived alcohol oxidase or glyceraldehyde-3-phosphatedehydrogenase. Examples of heterologous genes include: enzyme genes suchas acidic phosphatase gene, α-amylase gene and α-galactosidase gene;interferon genes such as interferon α gene and interferon γ gene;interleukin (IL) genes such as IL1 and IL2; cytokine genes such aserythropoietin (EPO) gene and granulocyte colony stimulating factor(G-CSF) gene; growth factor genes; and antibody genes. These genes maybe obtained by any procedures.

To utilize the invention efficiently, a gene encoding a glycoproteinproduced by a mammal cell, particularly human cell, can be used. Thatis, since the object of the invention is to produce a glycoprotein whichhas the same or similar sugar chain structure as that of mammalsparticularly human, the invention is effectively applied to theglycoprotein which has a sugar chain structure on the protein molecule,and additionally to useful physiologically active proteins includingantibodies. An antibody has been used as a medicament for many years.The antibody, however, was from an origin other than a human and so itcauses the production of an antibody against the administered antibodyitself. Accordingly, multiple administrations cannot be conducted, soits use is limited. In recent years, humanized antibody in which theamino acid sequence except the antigen-binding site is replaced by asequence of human antibody, has been prepared. Furthermore, a mouseproducing human antibody into which human antibody gene has beentransferred has been created. Complete human antibody is now availableand the use of an antibody as drug has prevailed quickly. Theseantibodies can be produced by hybridomas or by cultured cells such asCHO cell, which comprise a transfer gene encoding an antibody, howeverthere are many problems in respect of productivity, safety, etc. Undersuch a circumstance, production of antibodies using yeast is expected,because the above problems may be overcome by the use of yeast. In thiscase, as the antibody molecule is a glycoprotein to which N-type sugarchains are attached at two or more sites in each heavy chain, and whenthe antibody is produced with yeast, sugar chains peculiar to yeast areattached thereto. These sugar chains have antigenicity by themselves asmentioned above, and/or an action to decrease physiological activity.Hence, when the antibody produced with yeast is used as a medicament,the conversion of the sugar chain to a mammalian type is unavoidable.

In the meantime, the method for preparing antibodies with high ADCCactivity has been reported, which method comprises removal ofα-1,6-fucose attached to GlcNAc on the side of the reduced terminus of asugar chain (PCT/JP00/02260). Although α-1,6-fucosyl transferase gene(FUT8) is known as a gene involved in addition of α-1,6-fucose, thisgene is present ubiquitously in animal cells, and unless the cellsdeficient in this enzyme activity or the cells in which this gene isartificially disrupted are used, part of the prepared antibody isinevitably attached with α-1,6-fucose.

On the contrary, since the yeast generally has no synthetic systems offucose and α-1,6-fucosyl transferase gene (FUT8), glycoproteins freefrom α-1,6-fucose can be produced without artificial gene disruption.So, highly active antibodies could be naturally produced.

While there is a report on high production of antibody fragments such asFab and ScFv in yeast, there is almost no report on high production of afull-length antibody. Since antibody fragments such as Fab and ScFv donot comprise the Fc domain which exists in the heavy chain of anantibody, they have neither antibody-dependent cellular cytotoxicity(ADCC) nor complement-dependent cytotoxicity (CDC), which is aphysiological activity peculiar to an antibody, and their use as drug isrestricted. The antibody has 14 disulfide (S—S) linkages in total, andit is presumed that the reason why full-length antibody cannot be highlyproduce within a yeast cell is due to that the antibody molecule cannotappropriately fold. Although this cause is not clear, it cannot bedenied that the phenomenon may possibly be caused by difference in thestructure of N-type sugar chain attached to the antibody heavy chain.So, use of the yeast of the invention producing mammalian sugar chainsmay enable the efficient production of an antibody molecule havingsuitable conformation. Probably, functional antibody may also be highlyproduced by introducing Protein Disulfide Isomerase (PDI), a moleculechaperon. In addition, according to the invention, it is possible toproduce either an intact antibody molecule or other antibody fragmentsas mentioned above, or other antibody fragments as long as it has adesired function. The antibody is not particularly limited, butpreferred antibody includes a humanized antibody in which anantibody-binding site of another mammalian antibody is introduced into amammalian, particularly preferably human type framework, or a humanantibody. Although not limited particularly, the antibody to beexpressed is preferably in the class of IgG and more preferably in thesubclass of IgG1.

When a heterologous protein is produced by the gene recombinanttechnology, it is sometimes degraded by a protease in the host. In sucha case, the production of the protein of interest decreases,heterogeneous proteins generate, and the purification of the proteinbecomes difficult due to the contamination of proteolysis products.

In order to circumvent these problems, such a culture method that theactivity of a protease degrading the desired protein is inhibited hasbeen studied, for example, a method of adjusting the pH of a medium forculturing a recombinant cell to inhibit a protease activity. However,this method will affect the growth of host yeast which expresses acertain type of heterologous protein, and is effective only for thedegradation of the protein outside the cell.

There is an example which increased the production of cell proteinspresent inside and outside the cell by using a protease deficient strainin which proteinase A and proteinase B have been inactivated inSaccharomyces cerevisiae, Pichia pastoris, or Candida boidinii (JapanesePatent Publication (Kohyo) No. 6-506117A (1994), Weis, H. M. et al.,FEBS Lett., 377, 451 (1995), Inoue, K. et al., Plant Cell Physiol., 38(3), 366 (1997), and Japanese Patent Publication (Kokai) No.2000-78978).

Proteinase A and proteinase B are proteases located in the vacuole andare encoded by PEP4 gene and PRB1 gene, respectively. According toresearches on yeast Saccharomyces cerevisiae, proteinase A andproteinase B activate themselves and other proteases such ascarboxypeptidase Y (vandenHazel, H. B. et al., YEAST, 12, 1 (1996)).

In the meantime, Yapsin is a protease which exists widely in the Golgiapparatus and cell membrane, and according to researches onSaccharomyces cerevisiae, it was isolated as a homologue of the proteinencoded by KEX2 gene known as a processing enzyme of α-factor. To date,genes of Yapsin1 (Aspartic proteinase 3, YAP3), Yapsin2 (Asparticproteinase MKC7), Yapsin3, Yapsin6, Yapsin7, etc. are known(Egel-Mitani, M. et al., Yeast 6 (2), 127-137 (1990); Komano, H. et al.,Proc. Natl. Acad. Sci. U.S.A. 92(23), 10752-10756 (1995); andSaccharomyces Genome Database (SGD)). Of them, Yapsin1 is encoded byYPS1 gene.

An example in which the production of cell proteins present inside andoutside the cell was increased by using a protease deficientSaccharomyces cerevisiae strain in which Yapsin1 has been inactivated isknown (M. Egel-Mitani et al., Enzyme and Microbial Technology, 26, 671(2000); Bourbonniais, Y. et al., Protein Expr. Purif, 20, 485 (2000)).

Ogataea minuta strains of the invention deficient in PEP4 gene, PEP4PRB1gene or PEP4PRB1YPS1 gene, whose protease activities have been reduced,maintain an ability to grow themselves equivalent to the wild strainunder culture conditions of using a nutrition medium, and are thus verygood hosts for the production of heterologous proteins. Therefore, theabove-mentioned yeasts can efficiently produce heterologous proteins,such as an antibody highly susceptible to protease, due to suppressingthe degradation of the yeasts.

8. Construction of Expression Cassette for Heterologous Gene

The expression system useful for production of proteins can be preparedby various methods. A protein expression vector comprises at least apromoter area, a DNA encoding the protein, and the transcriptionterminator area in the direction of the reading frame of transcription.These DNAs are arranged as related operably to each other so that theDNA encoding the desired glycoprotein may be transcribed to RNA.

The high expression promoter which can be used in the invention ispreferably a methanol-inducible expression promoter, and includes, forexample, alcohol oxidase (AOX) gene promoter of Ogataea minuta,dihydroxyacetone synthase (DAS) gene promoter of Ogataea minuta, formatedehydrogenase (FDH) gene promoter of Ogataea minuta, etc.

The constitutive expression promoter includes glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene promoter of Ogataea minuta,phosphoglycerokinase (PGK) gene promoter of Ogataea minuta, etc.

The transcription terminator may be the sequence that has an activity tocause the termination of the transcription directed by the promoter, andmay be identical to or different from the promoter gene.

According to one aspect of the invention, we (1) obtained the nucleotidesequences of an Ogataea minuta alcohol oxidase(AOX) gene as amethanol-inducible expression cassette and a glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene as a constitutive expression cassette, alongwith their promoters and terminators, (2) isolated the promoters andterminators, (3) constructed expression vectors, and (4) used theexpression vectors of the invention to prepare transformed cells, andconfirmed that when expressed in the transformed cells, heterologousgenes are expressed in the same manner as the genes from Ogataea minuta.The expression cassette of a heterologous gene using the promoter andterminator for the alcohol oxidase (AOX) gene will be described below asan example.

8-1 Cloning of Alcohol Oxidase (AOX) Gene

In order to obtain the expression cassette of the invention, alcoholoxidase (AOX) gene was cloned at first. As the starting material, yeastsuch as Ogataea minuta strain IFO 10746 is exemplified. Cloning of thegene can be performed by a method as mentioned above.

8-2 Isolation of Promoter and Terminator Areas

Promoter and terminator areas can be cut out with a restrictionenzyme(s) but generally a convenient restriction site does notnecessarily exist at a suitable position. Accordingly, the nucleotidesequence may be cleaved in order from restriction sites in the codingarea toward the promoter area by an endonuclease, thereby to find aclone deleted until the suitable position. Recently, a primer with arestriction enzyme recognition site at the end has been used to beeasily able to amplify and obtain desired promoter and terminator areasby PCR.

It is also possible to chemically synthesize those areas, oralternatively, to make semi-synthesized promoter and terminator by useof both a DNA whose partial area is chemically synthesized and which isthen cloned using the partial DNA, and a restriction enzyme site(s).

The sequence comprising a promoter area or a terminator area isillustrated in SEQ ID NO:79 or in SEQ ID NO:80, respectively. They are,however, not to be limited to the specific sequences, and the nucleotidesequences thereof may be modified by deletion, insertion, substitution,addition, or the like, as long as they essentially hold transcriptionactivity.

Modification of the nucleotide sequences can be performed by any knownmutagenesis method (e.g., by the method using TAKARALA LA PCR in vitroMutagenesis kit, TAKARA SHUZO CO., LTD., Japan), or the like. When thepromoter area is deleted widely, this deletion may appropriately beconducted by PCR using a commercially available kit for deletion (e.g.,Deletion kit for kilo sequences of TAKARA SHUZO CO., LTD.).

8-3 Construction of Expression Vector

The expression vector of the invention can be obtained by inserting AOXpromoter, a heterologous structural gene, an AOX terminator, a markergene and a homologous area into an appropriate vector. Examples of thevector used for this purpose include, but are not limited to, E. coliplasmid vectors such as the above-mentioned pBR system, pUC system andBluescript system. Inserting the components of the expression vectorinto a vector can easily be carried out by those skilled in the art withreference to the description of Examples as described below or byconventional techniques. Those skilled in the art can determine theselectable marker gene and the homologous area easily. Examples of themarker gene include antibiotic resistance genes such as theabove-mentioned G-418 and hygromycin resistant genes, and auxotrophycomplementing genes such as URA3, ADE1 (phosphoribosyl-amino-imidazolesuccinocarboxamide synthase), HIS3 (imidazole-glycerol-phosphatedehydratase), LEU2 (3-isopropylmalate dehydrogenase) genes.

DNA encoding a secretion signal sequence which functions in a yeast cellmay be added to a heterologous structural gene. Since this expressionsystem allows production and secretion of a glycoprotein out of the hostcell, the desired glycoprotein can easily be isolated and purified. Thesecretion signal sequence includes secretion signal sequences ofSaccharomyces cerevisiae α-mating factor (α-MF), Saccharomycescerevisiae invertase (SUC2), human α-galactosidase, human antibody lightchains, etc.

The constructed expression vector is a chromosome integration typevector, and the desired gene is incorporated by being integrated ontothe chromosome. In the case of an auxotrophic marker type vector, a partof the marker gene is cleaved by a restriction enzyme(s) to form asingle stranded marker gene. Then the transformation is performed andthe vector is generally integrated into a part of the allele on thechromosome. In the case of a drug resistance marker, no allele exists,and so the expression promoter or terminator area is cleaved by arestriction enzyme(s) to form a single stranded promoter or terminator.Then the transformation is performed and the vector is generallyintegrated onto the above-mentioned part on the chromosome. Once thegene is integrated, it exists on a chromosome, and maintained stably.

8-4 Use of Expression Vector

The expression vector using the AOX promoter of the invention iseffective not only for expression of α-1,2-mannosidase gene andheterologous genes of interest but also for expression of other genes.By using expression vectors to which different types of selectablemarkers have been attached, the vectors can be transferred sequentiallyinto a yeast cell, and high expression of plural genes can be achieved.

For example, the yeast is not a host which originally generates asignificant amount of secreted proteins, when compared with mold or thelike. Thus, it is expected that the yeast bears no complete secretionmechanism. In fact, as mentioned above, the productivity of an antibodyin yeast is originally low.

Therefore, in order to enhance secretion efficiency, it is effectivethat a molecule chaperon or the like is introduced to attain highexpression.

9. Production of Glycoprotein Having Mammalian Type Sugar Chain

To produce glycoproteins having the above-mentioned sugar chains from aheterogeneous organism, the above-mentioned yeast mutant strain is usedas a host, and a gene in which a heterologous gene (e.g., cDNA) isligated downstream of a promoter and can be expressed in theabove-mentioned yeast, is prepared. The gene is integrated into theabove-mentioned yeast host by homologous recombination or inserted intoa plasmid to carry out transformation of the above-mentioned host. Thethus prepared transformant of the above-mentioned host is cultured byknown methods. The glycoprotein, which is encoded by the heterologousgene, produced intracellularly or extracelluraly is collected andpurified, thereby obtaining the glycoprotein.

The above-mentioned mammalian type sugar chain producing yeast mutantstrain maintains an ability to grow itself almost equivalent to the wildyeast strain, and this yeast mutant can be cultured by conventionalmethods as commonly used for culture of yeast. For example, thesynthesized medium (containing carbon source, nitrogen source, mineralsalts, amino acids, vitamins, etc.) supplemented with variousculture-medium ingredients as supplied from Difco and free from aminoacids as supplied by a marker required for duplication and maintenanceof the plasmid can be used (Scherman, Methods Enzymol., 194, 3-57(1991)).

The culture medium for expression of a heterologous gene by anexpression vector which is controlled by a methanol-inducible promoterto produce the desired gene expression product may contain a compoundwhich has an oxygen atom(s) or a nitrogen atom(s) and at least one C1substituent which binds to the atom. For example, methanol can be addedas the compound which has an oxygen atom, and at least one compoundselected from the group consisting of methylamine, dimethylamine,trimethylamine, and an ammonium compound with N-substituted methyl(e.g., choline) can be added as the compound having a nitrogen atom(s).

The medium may contain, in addition to methanol as the carbon source,one or more nitrogen sources such as yeast extract, tryptone, meatextract, casamino acid and ammonium salt, and mineral salts such asphosphate, sodium, potassium, magnesium, calcium, iron, copper,manganese and cobalt, and if necessary, trace nutrients such as varioustypes of vitamins and nucleotide, and appropriately carbohydratematerials for growth of yeast cells before the methanol induction.Specifically, the medium includes YPM medium (0.67% yeast nitrogen base,1% yeast extract, 2% peptone, 0.5% methanol), BYPM medium (0.67% yeastnitrogen base, 1% yeast extract, 2% peptone, 0.5% methanol, 0.1Mphosphate buffer pH 6.0), BM medium (0.67% yeast nitrogen base, 0.5%methanol, 0.1M phosphate buffer pH 6.0), etc.

The culture medium for expressing heterologous genes by an expressionvector, which is controlled by a constitutive expression promoter, toproduce a desired gene expression product includes culture mediumssuitable for cell growth. For example, synthesized media such as naturalculture media such as YPD medium (1% yeast extract, 2% peptone, 2%glucose) and SD medium (0.67% yeast nitrogen base, 2% glucose) can beused. Complementary nutrients may be supplemented in the above-mentionedmedia for yeast strains having an auxotrophic marker.

pH of the culture medium is suitably adjusted to 5.5 to 6.5. Culturetemperature is 15-30° C., preferably around 28° C. When the protein hasa complex conformation like an antibody, culturing at low temperature isdesirable in order to perform folding more efficiently within the cell.Culture time is about 24-1,000 hours, and culture can be conducted bymeans of standing culture, shaking culture, stirring culture, batchculture or continuous culture under aeration, or the like.

Conventional methods for isolation and purification of proteins can beused for isolating and purifying the expression product of aheterologous gene from the above-mentioned culture (i.e., culture brothor cultured cells).

For example, the cells may be collected by centrifugation after theculture, suspended in an aqueous buffer, and disrupted byultrasonicator, French press, Manton-Gaulin homogenizer, Dynomill or thelike, to obtain a cell-free extract. When the desired protein isproduced in the culture supernatant, the culture broth itself can beused. If necessary, a protease inhibitor may be added to the medium. Itis effective to use a protease deficient strain in order to suppressdegradation of the expression product of a heterologous gene. Purifiedpreparation or standard can be obtained by a conventional method forisolating and purifying proteins, from the supernatant obtained bycentrifugation of the cell-free extract or supernatant. Specifically,the purification can be conducted by using: for example, removal ofnucleic acids by protamine treatment; precipitation by fractionatingwith ammonium sulfate, alcohol, acetone added; anion exchangechromatography using resins such as DEAE Sepharose and Q Sepharose;cation exchange chromatography using resins such as S-Sepharose FF(Pharmacia); hydrophobic chromatography using resins such asbutylsepharose and phenylsepharose; gel filtration using molecularsieves; chelate columns such as His Bind resin (Novagen); affinitychromatography using resins such as Protein A Sepharose, specificdye-adsorbed resins such as Blue Sepharose; or lectin columns such as aConA Sepharose; reverse phase chromatography; chromatofocusing; andelectrofocusing; electrophoresis using polyacrylamide gel, singly or incombination, thereby to obtain the purified preparation or standard.However, the above-mentioned culture and purification methods arespecific examples and are not limited thereto.

The amino acid sequence of the purified gene product can be identifiedby the known amino acid analyses, such as the automated amino acidsequencing using the Edman degradation method.

EXAMPLES

The invention will now be described in detail with reference to specificexamples. These are for illustrative purposes only, and are not intendedto be limiting in any way the scope of the invention. The plasmids,enzymes such as restriction enzymes, T4 DNA ligase, and other substancesare all commercially available and can be used by conventional methods.Manipulations used in DNA cloning, sequencing, transformation of hostcells, culture of transformed cells, harvest of enzymes from resultantcultures, purification, etc. are also well known to those skilled in theart or can be known from the literature.

The restriction sites in restriction maps of various types of genes areshown by the following abbreviation. Ac; AccI, Ap; ApaI, Bl; BalI, Bm;BamHI, Bg; BglII, Bt; BtgI, Bw; BsiWI, Cl; ClaI, RI; EcoRI, RV; EcoRV,TI; EcoT22I, Hc; HincII, Hd; HindIII, Kp; KpnI, Nd; NdeI, Nh; NheI, Nt;NotI, Pf; PflMI, Pm; PmaCI, Ps; PstI, Sc; SacI, SI; SalI, Sm; SmaI, Sp;SpeI, Sh; SphI, Su; StuI, St; StyI, Xb; XbaI, and Xh; XhoI.

Example 1

Selection of Methylotrophic Yeast Suitable for Production of MammalianType Sugar Chain

To obtain a mammalian type sugar chain producing yeast usingmethylotrophic yeast, it is necessary to clone and inactivate a sugarchain synthesizing gene peculiar to the methylotrophic yeast. The sugarchain structure differs largely with the type of the yeast, as describedabove. In other words, the enzyme and gene involved in the biosynthesisof sugar chain also differ depending on the type of the yeast.Accordingly, when intending to disrupt the gene involved in thebiosynthesis of sugar chain to remove the sugar chain peculiar to theyeast, the first thing to do is to isolate the gene. As such isolation,however, requires a large number of steps, we decided to select amethylotrophic yeast, which requires the smallest possible number ofisolation steps. The selection of strains suitable for the isolation wasmade using NMR data on the cell wall of yeast as an indication ofselection (FIG. 3) (P. A. J. Gorin et al. (eds), Advanced inCarbohydrate Chemistry and Biochemistry, Vol. 23, 367-417 (1968)).Specifically, in a primary selection, strains suitable for isolationwere selected, which had an α-1,2-mannoside linkage-related signal ataround 4.3 ppm as a main peak but neither a α-1,3-mannosidelinkage-related signal at around 4.4 ppm nor any signals at 4.5 ppm orlarger. Then a secondary selection was made by extracting N-linked sugarchains from mannoprotein on the surface of the cells from the yeaststrains and analyzing the extracted sugar chains by α-1,2-mannosidasedigestion and HPLC. The methylotrophic yeast for the secondary selectionwere Candida succiphila IFO 1911 and Ogataea minuta IFO 10746. At thesame time, both of Saccharomyces cerevisiae having α-1,3-mannosidelinkage at unreduced termini of sugar chains, and Candida boidinii ATCC48180 which is a methylotrophic yeast having a peak at 4.5 ppm or largeron the above NMR data, were also analyzed as controls.

Fifty ml of YPD medium containing the above strains was put into a 500ml Sakaguchi flask, and cultured at 30° C. for 24-48 hours, and cellswere harvested from the culture by centrifugation, suspended in 10 ml of100 mM sodium citrate buffer (pH 7.0) and heated in autoclave at 121° C.for 1 hour. After cooling, the suspension was centrifuged to collect thesupernatant, 10 ml of water was added to the solid matter, and a mixturewas heated in the same manner as above and centrifuged to collect thesupernatant. The combined cell extracts were poured into 3 volumes ofethanol. The resultant white precipitate was dried, which was thendissolved in concanavalin A (ConA) column buffer (0.1 M sodium phosphatebuffer containing 0.15 M sodium chloride, 0.5 mM calcium chloride (pH7.2)), applied to a ConA-agarose column (0.6×2 cm, Honen Corporation),washed with ConA column buffer, and eluted with ConA column buffercontaining 0.2 M α-methylmannoside. Concanavalin A is a lectin that hasan affinity for sugar chains containing two or more α-D-mannose residueswhose C-3, C-4 and C-6 hydroxyl groups remain unsubstituted, and thecolumn with immobilized lectin enables the separation of mannan proteinfrom glucan, chitin and the like, which are yeast cell wallpolysaccharides (Peat et al. J. Chem. Soc., 29 (1961)). The resultantfraction was dialyzed and freeze-dried to yield mannan protein.

Then, the obtained mannan protein was treated with enzyme to cut outAsn-linked sugar chains. Specifically, the freeze-dried standard wasdissolved in 100 μl of N-glycosidase F buffer (0.1 M Tris-HCl buffercontaining 0.5% SDS, 0.35% 2-mercaptoethanol (pH 8.0)) and boiled for 5minutes. After cooling the boiled solution to room temperature, 50 μl of7.5% Nonidet P-40, 138 μl of H₂O and 12 μl of N-glycosidase F(Boehringer Ingelheim) were added and treated at 37° C. for 16 hours.After desalting with a BioRad AG501-X8 column, the equal amount ofphenol:chloroform (1:1) was added and vigorously shaken to remove thedetergent and proteins, to yield a sugar chain preparation.

To fluorescence-label (pyridylamination; referred to as PA) the obtainedsugar chains, the following were carried out. After concentrating thesugar chain preparation to dryness, 40 μl of a coupling agent (552 mg of2-aminopyridine dissolved in 200 μl of acetic acid) was added, sealed,and treated at 90° C. for 60 minutes. After cooling to room temperature,140 μl of a reducing agent (200 mg of borane-dimethylamine complexdissolved in 50 μl of H₂O and 80 μl of acetic acid) was added, sealed,followed by treating at 80° C. for 80 minutes. After reaction, 200 μl ofaqueous ammonia was added, the equal amount of phenol:chloroform (1:1)was added and vigorously shaken to recover the water layer thatcontained PA-oligosaccharides. A series of the steps was repeated 7times to remove unreacted 2-aminopyridine. The supernatant was filteredthrough a 0.22 μm filter to yield a PA-oligosaccharide preparation.

The obtained sugar chains were cleaved with Aspergillus saitoiα-1,2-mannosidase (SEIKAGAKU CORPORATION, Japan) and then analyzed byHPLC. HPLC using an amide column enables PA-oligosaccharides to beseparated depending on the chain length. The HPLC conditions were asfollows.

Column: TSK-Gel Amido-80 (4.6×250 mm, TOSOH CORPORATION, Japan)

Column temperature: 40° C.

Flow rate: 1 ml

Elution conditions: A: 200 mM triethylamine acetate pH 7.0+65%acetonitrile

-   -   B: 200 mM triethylamine acetate pH 7.0+30% acetonitrile    -   Linear gradient of 0 minute A=100% and 50 minutes A=0%

Excitation wavelength: 320 nm

Fluorescence wavelength: 400 nm

The results are shown in FIG. 4. The results revealed that N-linkedsugar chains derived from Ogataea minuta and Candida Succiphila weredegraded to small molecules of Man5 or Man6 by α-1,2-mannosidasetreatment, and thus suggested that sugar chain mutants (Man5 producingstrains) corresponding to och1, mnn1 and mnn4 in Saccharomycescerevisiae could be prepared by inactivation of OCH1 gene and expressionof α-1,2-mannosidase. On the other hand, for Candida boidinii, sugarchains remained undegraded at a considerably high rate. This is possiblydue to the linkage of a unit other than α-1,2-mannosidic linkage at theterminus of the sugar chains. Similarly, for Saccharomyces cerevisiae asthe control, there existed sugar chains undegraded, because possibleaddition of α-1,3-mannose resulting from the action of MNN1 gene.

Example 2

Cloning of glyceraldehyde-3-phosphate Dehydrogenase (GAP) Gene ofOgataea minuta

The GAP gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(2-1) Preparation of Probe

Oligonucleotides comprising nucleotide sequences corresponding to thefollowing amino acid sequences conserved in glyceraldehyde-3-phosphatedehydrogenases from Saccharomyces cerevisiae (GenBank accession number;P00359) and from Pichia pastoris (GenBank accession number; Q92263):

AYMFKYDSTHG; (SEQ ID NO: 1) and DGPSHKDWRGG (SEQ ID NO: 2)were synthesized as follows.

(SEQ ID NO: 3) PGP5; 5′-GCNTAYATGTTYAARTAYGAYWSNACNCAYGG-3′ (SEQ ID NO:4) PGP3; 5′-CCNCCNCKCCARTCYTTRTGNSWNGGNCCRTC-3′

The primer PGP5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence AYMFKYDSTHG, and the primerPGP3 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence DGPSHKDWRGG.

Chromosomal DNA was prepared from the cells of Ogataea minuta IFO 10746,which were cultured until stationary phase in YPD medium (comprising 1%yeast extract, 2% peptone, 2% glucose, pH 6.0), by means of potassiumacetate method (Methods in yeast genetics (1986), Cold Spring HarborLaboratory, Cols Spring Harbor, N.Y.).

PCR by Ex Taq polymerase (TAKARA SHUZO CO., LTD., Japan) ((94° C. for 30seconds, 50° C. for 1 minute and 72° C. for 45 seconds)×25 cycles) wascarried out using the obtained chromosomal DNA of Ogataea minuta IFO10746, as a template, and primers PGP5, PGP3. An amplified DNA fragmentof approximately 0.5 kb was recovered and cloned using TOPO TA CloningKit (Invitrogen). Plasmid DNA was isolated from the obtained clones andsequenced using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit(Applied Biosystems). For a DNA insert of the plasmid, a clone wasselected, which had a nucleotide sequence encoding an amino acidsequence having a high homology with the amino acid sequences for GAPgenes from Saccharomyces cerevisiae and Pichia pastoris. The 0.5-kb DNAinsert was recovered after EcoRI digestion of the plasmid and agarosegel electrophoresis.

(2-2) Construction of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes and subjected to 0.8% agarose gelelectrophoresis. The separated DNA was transferred to Hybond N+ nylonmembrane (Amersham). The DNA fragment obtained in Example (2-1) wasradiolabeled using Megaprimar DNA Labeling System (Amersham) andsubjected to Southern analysis. The hybridization was carried out byconventional procedure (Molecular cloning 2^(nd) edn., ed. Sambrook, J.,et al., Cold Spring Harbor Laboratory U.S.A., 1989). The resultssuggested that there existed a GAP gene in the HindIII-EcoRV fragment ofapproximately 6 kb. Then, to clone the DNA fragment, a library wasconstructed. The chromosomal DNA of Ogataea minuta was cleaved withHindIII and EcoRV and subsequently electrophoresed on agarose gel, andthe approximately 6-kb DNA fragment was recovered from the gel. Therecovered DNA fragment was ligated with HindIII- and HincII-cleaved pUC118 and then transformed into Escherichia coli DH5 α strain by theHanahan method (Gene, 10, 63 (1980)) to obtain a library.

Approximately 4,000 clones were screened by colony hybridization usingthe above described DNA fragment as a probe. A clone bearing plasmidpOMGP1 was selected from the 11 positive clones obtained.

(2-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the HindIII-BamHI region of the plasmidpOMGP1 (FIG. 5) was determined by deletion mutant and primer walkingmethod using Double-Stranded Nested Deletion Kit (Pharmacia). Thenucleotide sequence represented by SEQ ID NO:5 was determined byaligning the obtained nucleotide sequences.

In the nucleotide sequence of SEQ ID NO:5 there existed an open readingframe of 1,011 bp, starting at position 1,492 and ends at position2,502. The homology studies between the amino acid sequence (SEQ IDNO:6) deduced from the open reading frame and theglyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiaeor Pichia pastoris showed that 77% or 81% of amino acids wererespectively identical between them.

Example 3

Construction of Expression Cassette Using GAP Gene Promoter andTerminator

An expression cassette for transferring foreign genes was constructedbetween the GAP gene promoter (SEQ ID NO:7) and terminator (SEQ ID NO:8) of Ogataea minuta. A 3.2-kb HindIII-BamHI fragment was isolated frompOMGP1 described in Example 2-2 and inserted into the HindIII-BamHI ofpBluscript II SK−. The obtained plasmid was named pOMGP2 (FIG. 5). A3-kb HindIII-KpnI fragment was isolated from the pOMGP2 and the EcoRIsite was inserted into the HindIII-KpnI of blunt-ended pUC19. Theresultant plasmid was named pOMGP3 (FIG. 5). To transfer SalI andEcoT22I sites between the GAP gene promoter and terminator, the primers:

 5′-GTTTGAATTCACTCAATTAACATACACAAATACAATACAAAGTCGACAAAAA (SEQ ID NO: 9)ATGCATGTGGATAGATGACCAATGGCCTCTTTAAGTAAACATTTCGTTTTGAATAT ATTTC-3′, and 5′-TTTTTACTAGTACGGTACCGCTCGAATCGACACAGGAG-3′ (SEQ ID NO: 10)were synthesized. These primers were used to carry out PCR using thepOMGP2 as a template ((94° C. for 30 seconds, 55° C. for 1 minute and72° C. for 45 seconds)×20 cycles)). An amplified DNA fragment ofapproximately 0.6 kb was recovered and cloned using TOPO TA Cloning Kit.An inserted DNA fragment of 0.6 kb was isolated as an EcoRI-KpnIfragment and inserted into the EcoRI-KpnI of the pOMGP3. The obtainedplasmid was named pOMGP4 (FIG. 5). The pOMGP4 comprises an expressioncassette controlled by GAP gene promoter and terminator, which cassetteallows foreign genes to transfer into SalI-EcoT22I.

Example 4

Construction of G418 Resistant Gene Expression Cassette

To perform the transformation comprising selection of an antibiotic G418resistant gene, a plasmid was constructed which comprised an expressioncassette of a G418 resistant gene (aminoglycoside phosphotransferasegene). A 1.1-kb G418 resistant gene isolated, as a XhoI-PstI fragment,from plasmid pUC4K (Amersham Pharmacia) was inserted into theSalI-EcoT22I of the pOMGP4 constructed in Example 3. The resultantplasmid was named pOMKmR1.

Example 5

Cloning of orotidin-5′-phosphate Decarboxylase (URA3) Gene of Ogataeaminuta

The URA3 gene was obtained from Ogataea minuta IFO 10746, and itsnucleotide sequence was determined.

(5-1) Preparation of Probe

Oligonucleotides having the nucleotide sequences corresponding to theamino acid sequences conserved in orotidin-5′-phosphate decarboxylasesfrom Saccharomyces cerevisiae (GenBank accession number; K02207) andPichia pastoris (GenBank accession number; AF321098):

GPYICLVKTHID; (SEQ ID NO: 11) and GRGLFGKGRDP (SEQ ID NO: 12)were synthesized as follows.

(SEQ ID NO: 13) PUR5; 5′-GGNCCNTAYATHTGYYTNGTNAARACNCAYATHGA-3′ (SEQ IDNO: 14) PUR3; 5′-GGRTCNCKNCCYTTNCCRAANARNCCNCKNCC-3′

The primer PUR5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence GPYICLVKTHID, and the primerPUR3 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence GRGLFGKGRDP.

PCR by primers PUR5 and PUR3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 30 seconds)×25 cycles). The amplified DNAfragment of approximately 0.6 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences of orotidin-5′-phosphatedecarboxylases from Saccharomyces cerevisiae and Pichia pastoris. The0.6-kb DNA insert was recovered after EcoRI cleavage of the plasmid andagarose gel electrophoresis.

(5-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in (5-1) as a probe by the method described inExample (2-2). The results suggested that there was present URA3 gene inthe HindIII fragment of approximately 4.5 kb. Then, to clone the DNAfragment, a library was constructed. The chromosomal DNA of Ogataeaminuta was cleaved with HindIII and electrophoresed on agarose gel, andthen the approximately 4.5-kb DNA fragment was recovered from the gel.The resultant DNA fragment was ligated with HindIII-cleaved pUC18 andthen transformed into Escherichia coli DH5 α strain to obtain a library.

Approximately 6,000 clones were screened by colony hybridization usingthe above described DNA fragment as a probe. A clone bearing plasmidpOMUR1 was selected from the 3 positive clones obtained.

(5-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the NotI-HindIII region of the plasmid pOMUR1(FIG. 6) was determined by deletion mutant and primer walking method toobtain a nucleotide sequence represented by SEQ ID NO:15.

In the nucleotide sequence of SEQ ID NO:15, there existed an openreading frame of 798 bp, starting at position 1,732 and ends at position2,529. The homology studies between the amino acid sequence (SEQ IDNO:16) deduced from the open reading frame and the orotidin-5′-phosphatedecarboxylase from Saccharomyces cerevisiae or Pichia pastoris showedthat 82% or 75% of amino acids were respectively identical between them.

Example 6

Preparation of Ogataea minuta URA 3 Knockout Mutant

An Ogataea minuta URA3 knockout mutant was prepared by the “pop-in,pop-out” method (Rothstein R., Methods Enzymol., 194 (1991)).

(6-1) Preparation of UR43 Gene Disruption Vector

A 3-kb NotI-KpnI fragment was isolated from the plasmid pOMUR1 (FIG. 6)described in Example (5-2) and inserted into the NotI-KpnI ofpBluescript II SK−. After cleaving the plasmid with NotI and StyI,plasmid pOMUM1 (FIG. 6) was obtained by blunt-end treatment andself-ligation. Primers 5′-ATGGAGAAAAAAACTAGTGGATATACCACC-3′ (SEQ IDNO:17) and 5′-CTGAGACGAAAAAGATATCTCAATAAACCC-3′ (SEQ ID NO:18) were usedto carry out PCR using plasmid pHSG398 (TAKARA SHUZO CO., LTD., Japan)as a template ((94° C. for 30 seconds, 55° C. for 1 minute and 72° C.for 45 seconds)×20 cycles)) to amplify part of chloramphenicol resistantgene. The 0.4-kb amplified DNA fragment was cleaved with SpeI and EcoRVand inserted into the SpeI-RcoRV of the pOMUM1. The obtained plasmid wasnamed pOMUM2.

The plasmid pOMKmR1, which contained the G418 resistant gene expressioncassette controlled by the GAP gene promoter and terminator as preparedin Example 4, was cleaved with HindIII, blunt-ended, and ligated with aKpnI linker. The G418 resistant gene expression cassette was isolated asa 3-kb KpnI fragment from the plasmid and transferred at KpnI of thepOMUM2. The obtained plasmid was named pDOMU1 (FIG. 6).

(6-2) Transformation

The pDOMU1 constructed in Example (6-1) was cleaved with SalI andtransformed into Ogataea minuta IFO 10746 by the electric pulse method.The transformants were precultured in YPD medium at 30° C. overnight,inoculated into 100 ml of YPD medium, and cultured at 30° C. for 8-16hours until logarithmic growth phase (OD₆₀₀=about 1.5). The cells wereharvested by centrifugation at 1400×g for 5 minutes, washed once with100 ml of sterilized ice-cooled water, then once with 40 ml ofsterilized ice-cooled water. Then the cells were suspended in 20 ml ofLC buffer (100 mM LiCl, 50 mM potassium phosphate buffer, pH 7.5) andshaken at 30° C. for 45 minutes, and then 0.5 ml of 1 M DTT was added tothe suspension and shaken for another 15 minutes. After washed with 80ml of ice-cooled STM buffer (270 mM sucrose, 10 mM Tris-HCl buffer, pH7.5, 1 mM MgCl₂), the cells were suspended in 320 μl of STM buffer. Thetransformation by the electric pulse method was performed with GenePulser (BIO-RAD). After mixing 50 μl of the cell suspension and 5 μl ofDNA sample, the mixture was put into a 0.2 cm disposable cuvette, and anelectric pulse was applied to the mixture under appropriate conditions(voltage: 1.0 to 1.5 kv, resistance: 200-800 Ω). After application ofthe pulse, 1 ml of ice-cooled YDP medium containing 1 M sorbitol wasadded and subjected to shaking culture at 30° C. for 4-6 hours. Afterthe culture, the cell liquid was applied on a YPD selection mediumcontaining 400-1000 μg/ml G418, and the plate was incubated at 30° C. toobtain transformant colonies.

To confirm that the URA3 gene was disrupted, the following primers weresynthesized (see FIG. 7 with regard to the position of each primer).

DU5; 5′-AGGAAGAAGAGGAGGAAGAGGAAGAAAC-3′ (SEQ ID NO: 19) DUC5;5′-CGATGCCATTGGGATATATCAACGGTGG-3′ (SEQ ID NO: 20) DU3;5′-CCGTGTTTGAGTTTGTGAAAAACCAGGGC- (SEQ ID NO: 21) 3′ DUC3;5′-TGTGGCGTGTTACGGTGAAAACCTGGCC-3′ (SEQ ID NO: 22)

PCR by primers DU5 and DUC5 was performed using the chromosomal DNAisolated from the transformant as a template ((94° C. for 30 seconds,60° C. for 1 minute and 72° C. for 1 minute)×25 cycles). As shown inFIG. 7, a 1.1-kb amplified DNA fragment was detected from the strainwhose URA3 locus had the plasmid integrated there into. After culturingthe selected strain in the YPD medium until stationary phase, a strainresistant to 5-fluoroorotic acid (5-FOA) was obtained in accordance withthe method described in a manual for experimental procedures (MethodsEnzymol., 154, 164 (1987)). PCR by primers DU5 and DU3 ((94° C. for 30seconds, 60° C. for 1 minute and 72° C. for 3 minutes)×25 cycles), PCRby primers DU5 and DUC5 ((94° C. for 30 seconds, 60° C. for 1 minute and72° C. for 1 minute)×25 cycles), and PCR by primers DU3 and DUC3 ((94°C. for 30 seconds, 60° C. for 1 minute and 72° C. for 1 minute)×25cycles), were performed using the chromosomal DNA isolated from the5-FOA resistant strain as a template. As shown in FIG. 7, in the strainin which G418 resistant gene was deleted and the ORF of URA3 gene wasreplaced with the chloramphenicol resistant gene region, a 2.6-kbamplified DNA fragment was detected by PCR using DU5 and DU3, a 1.1-kbamplified DNA fragment by PCR using DU5 and DUC5, and a 1.0-kb amplifiedDNA fragment by PCR using DU3 and DUC3, respectively. The yeast wasnamed Ogataea minuta strain TK1-3 (ura3A).

Example 7

Cloning of ADEI (phosphoribosyl-amino-imidazole succinocarboxamidesynthase) Gene from Ogataea minuta

The ADE1 gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(7-1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to the aminoacid sequences conserved in the ADE1 gene products from Saccharomycescerevisiae (GenBank accession number; M61209) and Candida maltosa(GenBank accession number; M58322):

FVATDRISAYDVIM; (SEQ ID NO: 23) and QDSYDKQFLRDWLT (SEQ ID NO: 24)were synthesized as follows.

(SEQ ID NO: 25) PADS5; 5′- TTYGTNGCNACNGAYMGNATHWSNGCNTAYGAYGTNATHATG-3′ (SEQ ID NO: 26) PAD3; 5′- GTNARCCARTCNCKNARRAAYTGYTTRTCRTANSWRTCYTG-3′

The primer PAD5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence FVATDRISAYDVIM, and the primerPAD3 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence QDSYDKQFLRDWLT.

PCR by primers PAD5 and PAD3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 1 minute)×25 cycles). The amplified DNAfragment of approximately 0.7 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences of the ADE1 genes fromSaccharomyces cerevisiae and Candida maltosa.

The 0.7-kb DNA insert was recovered after EcoRI cleavage of the plasmidand agarose gel electrophoresis.

(7-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in (7-1) as a probe by the method described inExample (2-2). The results suggested that there existed ADE1 gene in theapproximately 5 kb HindIII-BamHI fragment. Then, to clone the DNAfragment, a library was prepared. The chromosomal DNA of Ogataea minutawas cleaved with HindIII and BamHI and electrophoresed on agarose gel,and then the approximately 5-kb DNA fragment was recovered from the gel.The DNA fragment was ligated with HindIII- and BamHI-cleaved pBluescriptII SK− and then transformed into Escherichia coli strain DH5 α toprepare a library.

Approximately 6,000 clones were screened by colony hybridization usingthe above described DNA fragment as a probe. A clone bearing plasmidpOMAD1 was selected from the 9 positive clones obtained.

(7-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the EcoRV-SmaI region of the plasmid pOMAD1(FIG. 8) was determined by deletion mutant and primer walking method toobtain a nucleotide sequence represented by SEQ ID NO:27.

In the nucleotide sequence of SEQ ID NO:27, there existed an openreading frame of 912 bp, starting at position 939 and ends at position1,850. The homology studies between the amino acid sequence (SEQ IDNO:28) deduced from the open reading frame and the ADE1 gene productfrom Saccharomyces cerevisiae or Pichia pastoris showed that 69% or 74%of amino acids were respectively identical between them.

Example 8

Preparation of Ogataea minuta ADE1 Knockout Mutant

The ADE1 gene was disrupted by transformation using the URA43 gene ofOgataea minuta as a marker.

(8-1) Preparation of ADE1 Disruption Vector

As shown in FIG. 8, plasmid pDOMAD1 was prepared by replacingapproximately 70-bp region of the ADE1 structural gene by the URA3 gene.To obtain a uracil auxotrophic mutant again from ADE1 gene knockoutmutants, the URA3 gene having repetitive structures before and after thestructural gene was used as a marker. PCR by the primers:

5′-CCCCGAGCTCAAAAAAAAGGTACCAATTTCAGCTCCGACGCCGGAGCCCACT (SEQ ID No. 29)ACGCCTAC-3′; and 5′-GGGAAGCTTCCCCAGTTGTACACCAATCTTGTCGACAG-3′ (SEQ IDNo. 30)was performed using, as a template, the plasmid pOMUR1 having the URA3gene region as described in Example 5 ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 45 seconds)×20 cycles) to amplify theupstream region of the URA3 structural gene. The amplified DNA fragmentof approximately 0.8 kb was recovered, cleaved with SacI and HindIII,and inserted into the SacI-HindIII of the pUC18.

The 3.3-kb SacI-KpnI fragment isolated from the pOMUR1 was inserted intothe SacI-KpnI of the obtained plasmid. The resultant plasmid was cleavedwith KpnI, blunt-ended, and self-ligated. The obtained plasmid was namedpOMUR2 (FIG. 9). The pOMUR2 was cleaved with StyI, blunt-ended, andligated with a BglII linker. The obtained plasmid was named pROMU1. Inthe 3.3-kb DNA fragment obtained by cleaving the pROMU1 with BglII andHindIII, there existed approximately 0.8-kb repetitive sequences beforeand after the URA3 structural gene (FIG. 9).

PCR by the primers:

Dad1-5: 5′-AAAAAGCGGCCGCTCCCGGTGTCCCGCAGAAATCTTTATGCGTAGTCTT (SEQ ID NO:31) G-3′; and Dad1-3:5′-CCCCCGGATCCTTTTTTTTAAGCTTGTTGTACTCCTTCCATGCACTTCCGG (SEQ ID NO: 32)TGATG-3′((94° C. for 30 seconds, 50° C. for 1 minute and 72° C. for 1 minute)×20cycles), and

PCR by the primers:

(SEQ ID NO: 33) Dad2-5: 5′-TTTTCACCCCGTCAAGGATCCCTGAACAAGGCGAACACGACGAAAACA TTTCCCCCGAG-3′; and(SEQ ID NO: 34) Dad2-3: 5′-TTTTTGGGCCCACCTGGGTGAAGATTTGCCAGATCAAGTTCTCC-3′((94° C. for 30 seconds, 50° C. for 1 minute and 72° C. for 1 minute)×20cycles) were performed using, as a template, the plasmid pOMAD1 havingthe ADE1 gene region as described in Example 7. The amplified DNAfragments of approximately 0.7 kb and 1 kb were recovered and cleavedwith NotII and BamHI and with BamHI and ApaI, respectively. Both of theNotI-BamHI and BamHII-ApaI DNA fragments obtained were inserted into theNotI-ApaI of the pBluescript II SK−. The 3.3-kb BglII-HindIII fragmentisolated from the pROMU1 was inserted into the BamHI-HindIII of theobtained plasmid. The resultant plasmid was named pDOMAD1 (FIG. 8).(8-2) Transformation

The pDOMAD1 obtained in Example (8-1) was cleaved with ApaI and NotI andtransformed into Ogataea minuta strain TK1-3 (ura3Δ) obtained in Example(6-2) by the electric pulse method. Strains exhibiting ade1 traitproduce a red pigment, which is an intermediate metabolite in theadenine biosynthesis, and their colonies are dyed red. Thus, strainswhose colonies were dyed red compared with the transformants wereselected. To confirm that the ADE1 genes of these strains weredisrupted, the following primers were synthesized (see FIG. 10 withregard to the position of each primer).

DA5; 5′- (SEQ ID NO: 35) GATGCTTGCGCCTTCAACCACATACTCCTC-3′ DA3; 5′- (SEQID NO: 36) AAAAGTTCTTGCACAGCCTCAATATTGACC-3′ DOU5; 5′- (SEQ ID NO: 37)ATCGATTTCGAGTGTTTGTCCAGGTCCGGG-3′

PCR by primers DA5 and DOU5 was performed using the chromosomal DNAisolated from the transformant as a template ((94° C. for 30 seconds,60° C. for 1 minute and 72° C. for 2 minutes)×25 cycles). As shown inFIG. 10, a 1.6-kb amplified DNA fragment was detected from the strainwhose ADE1 locus had the plasmid integrated thereinto. After culturingthe selected strain in the YPD medium until stationary phase, a strainresistant to 5-fluoroorotic acid (5-FOA) was obtained. PCR by primersDA5 and DA3 was performed using the chromosomal DNA isolated from the5-FOA resistant strain as a template ((94° C. for 30 seconds, 60° C. for1 minute and 72° C. for 3 minutes)×25 cycles). As shown in FIG. 10, inthe strain in which URA3 gene was deleted, a 2.9-kb amplified DNAfragment was detected. The ura3Δ ade1Δ strain was named Ogataea minutastrain TK4-1.

Example 9

Cloning of OCH1 Gene from Ogataea minuta

The OCH1 gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(9- 1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to the aminoacid sequences conserved in OCH1 gene products from Saccharomycescerevisiae (GenBank accession number; P31755) and Pichia pastoris(Japanese Patent Publication (Kokai) No. 9-3097A):

PQH(R)I(V)WQTWKV; (SEQ ID NO: 38) and WYARRIQFCQW (SEQ ID NO: 39)were synthesized as follows.

POH5; 5′- (SEQ ID NO: 40) CCNCARCRYRTHTGGCARACNTGGAARGT-3′ POH3; 5′-(SEQ ID NO: 41) CCAYTGRCARAAYTGDATNCKNCKNGCRTACCA- 3′

The primer POH5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence PQH(R)I(V)WQTWKV, and theprimer POH3 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence WYARRIQFCQW.

PCR by primers POH5 and POH3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 30 seconds)×25 cycles). The amplified DNAfragment of approximately 0.4 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences of OCH1 gene products fromSaccharomyces cerevisiae and Pichia pastoris. The 0.4-kb DNA insert wasrecovered after EcoRI cleavage of the plasmid and agarose gelelectrophoresis.

(9-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (9-1) as a probe by the methoddescribed in Example (2-2). The results suggested that there existedOCH1 gene in the XbaI fragment of approximately 5 kb. Then, to clone theDNA fragment, a library was prepared. The chromosomal DNA of Ogataeaminuta was cleaved with XbaI and subjected to agarose gelelectrophoresis, and then the approximately 5-kb DNA fragment wasrecovered from the gel. The recovered DNA fragment was ligated withXbaI-cleaved pBluescript II SK− and then transformed into Escherichiacoli DH5 α strains to prepare a library.

Approximately 6,000 clones were screened by colony hybridization usingthe above described DNA fragment as a probe. A clone bearing plasmidpOMOC1 was selected from the 4 positive clones obtained.

(9-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the BglII-SpeI region of the plasmid pOMOC1(FIG. 11) was determined by deletion mutant and primer walking method toobtain a nucleotide sequence represented by SEQ ID NO:42.

In the nucleotide sequence of SEQ ID NO:42 there existed an open readingframe consisting of 1,305 bp, starting at position 508 and ends atposition 1,812. The homology studies between the amino acid sequence(SEQ ID NO:43) deduced from the open reading frame and themannosyltransferase OCH1 gene product from Saccharomyces cerevisiae orPichia pastoris showed that 42% or 29% of amino acids were respectivelyidentical between them. It remains unknown whether or not the Pichiapastoris-derived OCH1 gene disclosed in Japanese Patent Publication(Kokai) No. 9-3097A substantially encodes the OCH1 (α-1,6mannosyltransferase), or whether or not the same Pichia pastoris-derivedOCH1 gene has the functions of the OCH1 gene of Ogataea minuta describedin this Example and Examples 10 and 11. The reasons are that thehomology to the Pichia pastoris-derived OCH1 was 29% in amino acid, andthat it has not been studied whether the Pichia pastoris-derived OCH1has the activity of the Saccharomyces cerevisiae-derived OCH1 (α-1,6mannosyltransferase).

Example 10

Preparation of Ogataea minuta-derived OCH1 Knockout Mutant

The OCH1 gene was disrupted by transformation using the URA3 gene ofOgataea minuta as a marker.

(10-1) Preparation of OCH1 Gene Disruption Vector

Plasmid pDOMOCH1 was prepared by replacing approximately 0.5-kbBalI-SmaI region of the OCH1 gene by the URA3 gene (FIG. 11). To obtaina uracil auxotrophic mutant again from OCH1 knockout mutant, the URA3gene having repetitive structures before and after the structural gene,as described in Example (8-1), was used as a marker.

The 4.4-kb NotI-XbaI fragment was isolated from the pOMOC1 and insertedinto the NotI-XbaI of pBluescript II SK−. The obtained plasmid was namedpOMOC2. The pOMOC2 was cleaved with AccI and XhoI, blunt-ended, andself-ligated. The obtained plasmid was named pOMOC3. The pOMOC2 wascleaved with BalI, and ligated with a BamHI linker. The obtained plasmidwas named pOMOC2B (FIG. 11). The pOMOC3 was cleaved with SmaI, andligated with a HindIII linker. The obtained plasmid was named pOMOC3H(FIG. 11). The 3.3-kb BglII-HindIII fragment isolated from the pROMU1described in Example (8-1) was inserted into the BamHI-HindIII of thepOMOC2B. The 1.5-kb HindIII-ApaI fragment isolated from the pOMOC3H wasinserted into the HindIII-ApaI of the obtained plasmid. The resultantplasmid was named pDOMOCH1.

(10-2) Transformation

The pDOMOCH1 obtained in Example (10-1) was cleaved with ApaI and NotI,and transformed into Ogataea minuta TK1-3 strain (ura3Δ), which wasobtained in Example (6-2), and into Ogataea minuta TK4-1 strain (ura3ΔAdel1Δ), which was obtained in Example (8-2), by electric pulse method.The transformation was performed in accordance with the method describedin Example (6-2).

To confirm that the OCH1 genes of these strains were disrupted, thefollowing primers were synthesized (see FIG. 12 with regard to theposition of each primer).

DO3; 5′- (SEQ ID NO: 44) CCATTGTCAGCTCCAATTCTTTGATAAACG-3′ DOU5; 5′-(SEQ ID NO: 37) ATCGATTTCGAGTGTTTGTCCAGGTCCGGG-3′ DO5; 5′- (SEQ ID NO:45) ACACTTCCGTAAGTTCCAAGAGACATGGCC-3′ DO3-2; 5′- (SEQ ID NO: 46)TCACCACGTTATTGAGATAATCAAACAGGG-3′

PCR by primers DO5 and DOU5 was performed using the chromosomal DNAisolated from the transformant as a template ((94° C. for 30 seconds,60° C. for 1 minute and 72° C. for 3 minutes)×25 cycles). As shown inFIG. 12, a 2.4-kb amplified DNA fragment was detected in the strainwhose OCH1 locus had the plasmid integrated thereinto. After culturingthe selected strain in the YPD medium until stationary phase, a strainresistant to 5-fluoroorotic acid (5-FOA) was obtained. PCR by primersDO3 and DO5 ((94° C. for 30 seconds, 60° C. for 1 minute and 72° C. for3 minutes)×25 cycles) and PCR by primers DO5 and DOC3-2 ((94° C. for 30seconds, 60° C. for 1 minute and 72° C. for 1 minute)×25 cycles) wereperformed using the chromosomal DNA isolated from the 5-FOA resistantstrain as a template. As shown in FIG. 12, in the strain in which URA3gene was deleted, a 2.4-kb amplified DNA fragment was detected by thePCR using primers DO3 and DO5 and a 0.9 kb amplified DNA fragment by thePCR using primers DO5 and DOC3-2. The och1Δ ura3Δ strain obtained wasnamed Ogataea minuta TK3-A strain, and the och1Δ ura3Δ ade1Δ strain wasnamed Ogataea minuta TK5-3 strain.

Example 11

Isolation of Cell Surface Mannan Protein from Ogataea minuta OCH1Knockout Mutant and Structure Analysis of Sugar Chain Contained Therein

Structure analysis of sugar chains of cell surface mannan proteins wasperformed for Ogataea minuta OCH1 knockout mutant strainTK3-A and itsparent strain TK1-3. The preparation of PA-oligosaccharides wasperformed by the method described in Example 1.

The prepared sugar chains were cleaved with Aspergillus saitoiα-1,2-mannosidase (SEIKAGAKU CORPORATION, Japan). Analysis was performedby HPLC. HPLC on amide column enables PA-oligosaccharides to beseparated depending on the chain length. HPLC using a reverse-phasecolumn enables PA-oligosaccharides to be separated depending on thehydropholicity, thereby to identify sugar chain structures. The HPLCconditions were as follows.

1) Size Analysis by Amide Column

Column: TSK-Gel Amido-80 (4.6×250 mm, TOSOH CORPORATION, Japan)

Column temperature: 40° C.

Flow rate: 1 ml

Elution conditions: A: 200 mM triethylamine acetate pH 7.0+65%acetonitrile

-   -   B: 200 mM triethylamine acetate pH 7.0+30% acetonitrile    -   Linear gradient of 0 minute A=100% and 50 minutes A=0%        2) Structure Analysis by Reverse Phase Column

Column: TSK-Gel ODS80TM (4.6×250 mm, TOSOH CORPORATION, Japan)

Column temperature: 50° C.

Flow rate: 1.2 ml

Elution conditions: 100 mM ammonium acetate containing 0.15% n-butanolpH 6.0

The results are shown in FIG. 13. From the size analysis using an amidecolumn, it was confirmed that the TK1-3 strain as a parent strainproduced both Man5 and Man6 as shown in FIG. 13, whereas the TK3-Astrain, i.e., a ΔOCH1 strain, mainly produced Man5. Further, from thestructure analysis using a reverse phase column and the comparison withcommercially available standard sugar chains (TAKARA SHUZO CO., LTD.,Japan), it was found that Man6 of the TK1-3 strain was a sugar chainhaving the structural formula 1 below, Man5 of the TK1-3 strain a sugarchain having the structural formula 2 below, and Man5 of the TK3-Astrain a sugar chain having the structural formula 2 below.

From the above results, it was confirmed that the obtained gene wassubstantially Ogataea minuta OCH1 gene and that it was possible toprepare sugar chain mutants corresponding to the och1, mnn1 and mnn4strains in Saccharomyces cerevisiae in which α-1,2-mannosidase gene wasexpressed.

Example 12

Cloning of Proteinase A (PEP4) Gene of Ogataea minuta

The PEP4 gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(12-1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to thefollowing amino acid sequences conserved in PEP4 gene from Saccharomycescerevisiae (GenBank accession number; M13358) and Pichia angusta(GenBank accession number; U67173):

TNYLNAQY; (SEQ ID NO: 47) and KAYWEVKF (SEQ ID NO: 48)were synthesized as follows.

PPA5; 5′- (SEQ ID NO: 49) ACNAAYTAYYTNAAYGCNCARTA-3′ PPA3; 5′- (SEQ IDNO: 50) AAYTTNACYTCCCARTANGCYTT-3′

The primer PPA5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence TNYLNAQY, and the primer PPA3has a sequence complementary to the nucleotide sequence corresponding tothe amino acid sequence KAYWEVKF.

PCR by primers PPA5 and PPA3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 55° Cfor 1 minute and 72° C. for 1 minute)×25 cycles). The amplified DNAfragment of approximately 0.6 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences for PEP4 genes from Saccharomycescerevisiae and Pichia angusta. The 0.6-kb DNA insert was recovered afterEcoRI cleavage of the plasmid and agarose gel electrophoresis.

(12-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (12-1) as a probe by the methoddescribed in Example (6-2). The results suggested that there existedPEP4 gene in the approximately 6 kb BamHI fragment. Then, to clone theDNA fragment, a library was prepared. The chromosomal DNA of Ogataeaminuta was cleaved with BamHI and subjected to agarose gelelectrophoresis, and then the approximately 6-kb DNA fragment wasrecovered from the gel. The recovered DNA fragment was ligated withBamHI-cleaved pUC18 and then transformed into Escherichia coli strainDH5 α to prepare a library.

About 5,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMPA1 wasselected from the 8 positive clones obtained.

(12-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the NdeI-XbaI region of the plasmid pOMPA1(FIG. 14) was determined by deletion mutant and primer walking method toobtain a nucleotide sequence represented by SEQ ID NO:51.

In the nucleotide sequence represented by SEQ ID NO:51, there existed anopen reading frame of 1,233 bp, starting at position 477 and ends atposition 1,709. The homology studies between the amino acid sequence(SEQ ID NO:52) deduced from the open reading frame and the PEP4 fromSaccharomyces cerevisiae or Pichia angusta showed that 67% or 78% ofamino acids were respectively identical between them.

Example 13

Preparation of Ogataea minuta PEP4 Knockout Mutant

The PEP4 gene was disrupted by transformation using the URA3 gene ofOgataea minuta as a marker.

(13-1) Preparation of PEP4 Disruption Vector

As shown in FIG. 14, plasmid pDOMPA1 was prepared by replacing theapproximately 1.1-kb SmaI-XbaI region of the PEP4 structural gene by theURA3 gene. To obtain a uracil auxotrophic mutant again from PEP4knockout mutants, the URA3 gene having repetitive structures before andafter the structural gene was used as a marker. Plasmid was prepared bySacI cleavage, self-ligation, ClaI cleavage, and self-ligation of theplasmid pOMPA1 carrying the PEP4 gene region, as described in Example(12-2).

The obtained plasmid was cleaved with SmaI, ligated with a HindIIIlinker, cleaved with XbaI, blunt0ended, and ligated with a BglII linker.

The 3.3-kb BglII-HindIII fragment isolated from the pROMMI described inExample (8-1) was inserted into the BglII-HindIII of the obtainedplasmid. The resultant plasmid was named pDOMPA1 (FIG. 14).

(13-2) Transformation

The pDOMPA1 obtained in Example (13-1) was cleaved at SacI-ClaI, andthen transformed into the Ogataea minuta TK3-A strain (och1Δ ura3Δ) andthe Ogataea minuta TK5-3 strain (och1Δ ura3Δ ade1Δ) obtained in Example(10-2), by means of the electric pulse method.

The PEP4 knockout mutants were screened by subjecting the chromosomalDNAs of the obtained transformants to Southern analysis. Specifically,when cleaving the chromosomal DNAs of the host strain and thetransformants with BamHI and subjecting the cleaved chromosomal DNAs toSouthern analysis using the 4.8-kb SacI-ClaI fragment isolated from thepDOMPA1 (FIG. 14) as a probe, a band was detected at 6 kb in the hoststrain, while a band was detected at 9 kb in the knockout mutants. Afterculturing the knockout mutants in the YPD medium until stationary phase,a strain resistant to 5-fluoroorotic acid (5-FOA) was obtained. Thechromosomal DNA of the 5-FOA resistant strain was cleaved with BamHI andagain subjected to Southern analysis using the 4.8-kb SacI-ClaI fragmentisolated from the pDOMPA1 (FIG. 14) as a probe, and a strain wasselected from which the UR43 gene was deleted and in which a band wasdetected at 5.5 kb. The och1Δ pep4Δ ura3Δ strain obtained was namedOgataea minuta TK6 strain, and the och1Δ pep4Δ ura3Δ ade1Δ strain wasnamed Ogataea minuta TK7 strain.

Example 14

Cloning of PRB1 Gene of Ogataea minuta

The PRB1 gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(14-1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to thefollowing amino acid sequences conserved in PRB1 from Saccharomycescerevisiae (GenBank accession number; M18097) and Kluyveromyces lactis(GenBank accession number; A75534) and their homologues:

DG(L)NGHGTHCAG (SEQ ID NO: 53) GTSMAS (T) PHV (I) A (V) G (SEQ ID NO:54)were synthesized as follows.

PPB5; 5′- (SEQ ID NO: 55) GAYBKNAAYGGNCAYGGNACNCAYTGYKCNGG- 3′ PPB3; 5′-(SEQ ID NO: 56) CCNRCNAYRTGNGGNWSNGCCATNWSNGTNCC- 3′

The primer PPB5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence DG(L)NGHGTHCAG, and the primerPPB3 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence GTSMAS(T)PHV(I)A(V)G.

PCR by primers PPB5 and PPB3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 1 minute)×25 cycles). The amplified DNAfragment of approximately 0.5 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences for PRB1 genes from Pichiapastoris and Kluyveromyces lactis. The 0.5-kb DNA insert was recoveredafter EcoRI cleavage of the plasmid and agarose gel electrophoresis.

(14-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (14-1) as a probe by the methoddescribed in Example (2-2). The results suggested that there existedPRB1 gene in the BamHI fragment of approximately 5 kb. Then, to clonethe DNA fragment, a library was prepared. The chromosomal DNA of Ogataeaminuta was cleaved with BamHI and electrophoresed on agarose gel, andthen the approximately 5-kb DNA fragment was recovered from the gel. TheDNA fragment was ligated with BamHI-cleaved and BAP-treated pUC18 andthen transformed into Escherichia coli strain DH5 α to prepare alibrary.

About 6,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMPB 1 wasselected from the 2 positive clones obtained.

(14-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the BamHI-HindIII region of the plasmidpOMPB1 (FIG. 15) was determined by deletion mutant and primer walkingmethod to obtain a nucleotide sequence represented by SEQ ID NO:57.

In the nucleotide sequence of SEQ ID NO:57, there existed an openreading frame of 1,620 bp, starting at position 394 and ends at position2,013. The homology studies between the amino acid sequence (SEQ ID NO:)deduced from the open reading frame and the PRB1 gene product fromPichia pastoris or Kluyveromyces lactis showed that 47% or 55% of aminoacids were respectively identical between them.

Example 15

Preparation of Ogataea minuta PRB1 Knockout Mutant

The PRB1 gene was disrupted by transformation using the URA3 gene ofOgataea minuta as a marker.

(15-1) Preparation of PRB1 Gene Disruption Vector

As shown in FIG. 15, plasmid pDOMPB1 was prepared by replacing theapproximately 0.2-kb ClaI-SphI region of the PRB1 structural gene by theURA3 gene. To obtain a uracil auxotrophic mutant again from PRB1knockout mutants, the URA3 gene having repetitive structures before andafter the structural gene was used as a marker. The BamHI fragment wasisolated from the plasmid pOMPB1 having the PRB1 gene region asdescribed in Example (14-2) and inserted into pTV19ΔSph (i.e., pTV19which was cleaved with SphI, blunt-ended and self-ligated, and fromwhich SphI site was deleted), which had been cleaved with BamHI andtreated with BAP.

The 3.3-kb ClaI-SphI fragments isolated from the plasmid, as describedin Example (8-1), which were obtained by changing the BglII site of thepROMU1 to a ClaI site and changing the HindIII site of the pROMU1 to aSphI site, respectively, by linker ligation method, were inserted intothe ClaI-SphI of the obtained plasmid. The resultant plasmid was namedpDOMPB1 (FIG. 15).

(15-2) Transformation

The pDOMPB1 obtained in Example (15-1) was cleaved with BamHI andtransformed into the Ogataea minuta TK6 strain (och1Δ pep4Δ ura3Δ) andthe Ogataea minuta TK7 strain (och1Δ pep4Δ ura3Δ ade1Δ) obtained inExample (13-2) by electric pulse method.

The PRB1 knockout mutants were screened by subjecting the chromosomalDNAs of the obtained transformants to Southern analysis. Specifically,when cleaving the chromosomal DNAs of the host strain and thetransformants with BamHI and subjecting the cleaved chromosomal DNAs toSouthern analysis using the 5-kb BamHI fragment isolated from thepDOMPB1 (FIG. 15) as a probe, 5 kb band was detected in the host strain,while 8.5 kb band was detected in the knockout mutants. After culturingthe knockout mutants in the YPD medium until stationary phase, a strainresistant to 5-fluoroorotic acid (5-FOA) was obtained. The chromosomalDNA of the 5-FOA resistant strain was cleaved with BamHI and againsubjected to Southern analysis using the 5-kb BamHI fragment isolatedfrom the pDOMPB1 (FIG. 15) as a probe, and a strain was selected fromwhich the URA3 gene was deleted and for which 5 kb band was detected.The och1Δ pep4Δ prb1Δ ura3Δ strain obtained was named Ogataea minuta TK8strain, and the och1Δ pep4Δ prb1Δ ura3Δ ade1Δ strain was named Ogataeaminuta TK9 strain.

Example 16

Cloning of KTR1 Gene of Ogataea minuta

The KTR1 gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(16-1) Preparation of Probe

The amino acid sequences conserved in the KTR gene family fromSaccharomyces cerevisiae (Biochim. Biophys, Acta, (1999) Vol. 1426,p326) was extracted:

H(N)YDWV(T)FLND; (SEQ ID NO: 59) and YNLCHFWSNFEI, (SEQ ID NO: 60)and oligonucleotides having nucleotide sequences corresponding the aboveamino acid sequences were synthesized as follows.

(SEQ ID NO: 61) PKR5; 5′-MAYTAYGAYTGGRYNTTYYTNAAYGA-3′ (SEQ ID NO: 62)PKR3; 5′-ATYTCRAARTTNSWCCARAARTGRCANARRTTRTA-3′

The primer PKR5 has a sequence complementary to the nucleotide sequencescorresponding to the amino acid sequence H(N)YDWV(T)FLND, and the primerPKR3 has a sequence complementary to the nucleotide sequencescorresponding to the amino acid sequence YNLCHFWSNFEI.

PCR by primers PKR5 and PKR3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 1 minute)×25 cycles). The amplified DNAfragment of approximately 0.6 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. From the nucleotide sequence analysis for 60 clones, it wasconfirmed that total 4 types of gene fragments existed, all of which hada high homology with the amino acid sequences of the KTR1 gene familyfrom Saccharomyces cerevisiae. One clone was selected from the 60 clonesand the 0.6-kb DNA insert was recovered after EcoRI cleavage of theplasmid and separation by agarose gel electrophoresis.

(16-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes and subjected to Southern analysis usingthe DNA fragment obtained in Example (12-1) as a probe by the methoddescribed in Example (2-2). The results suggested that there existed theKTR1 gene in the SacI fragment of approximately 2 kb. Then, to clone theDNA fragment, a library was prepared. The chromosomal DNA of Ogataeaminuta was cleaved with SacI and subjected to agarose gelelectrophoresis, and then the approximately 2-kb DNA fragment wasrecovered from the gel. The DNA fragment was ligated with SacI-cleavedand BAP-treated pUC18 and then transformed into Escherichia coli strainDH5 α to prepare a library.

About 4,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMKR1 wasselected from the 2 positive clones obtained.

(16-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the SacI insert in the plasmid pOMKR1 (FIG.16) was determined by deletion mutant and primer walking method toobtain a nucleotide sequence represented by SEQ ID NO:63.

In the nucleotide sequence of SEQ ID NO:63, there existed an openreading frame of 1,212 bp, starting at position 124 and ends at position1,335. The homology studies between the amino acid sequence (SEQ IDNO:64) deduced from the open reading frame and the KTR1 or KRE2 geneproduct, as KTR family, from Saccharomyces cerevisiae, showed that 53%or 49% of amino acids were respectively identical between them

Example 17

Preparation of Ogataea minuta KTR1 Knockout Mutant

The KTR1 gene was disrupted by transformation using the URA3 gene ofOgataea minuta as a marker.

(17-1) Preparation of KTR1 Gene Disruption Vector

As shown in FIG. 16, plasmid pDOMKR1 was prepared by replacing the0.3-kb EcoRI-BglII region of the KTR1 structural gene by the URA3 gene.To obtain a uracil auxotrophic mutant again from KTR1 knockout mutants,the URA3 gene having repetitive structures before and after thestructural gene was used as a marker. The plasmid pOMKR1 carrying theKTR1 gene region as described in Example (16-2) was cleaved atHindIII-XbaI, blunt-ended, and ligated. The obtained plasmid was cleavedwith EcoRI and ligated with a HindIII linker.

The 3.3-kb BglII-HindIII fragment isolated from the pROMU1 as describedin Example (8-1) was inserted into the BglII-HindIII of the obtainedplasmid. The resultant plasmid was named pDOMKR1 (FIG. 16).

(17-2) Transformation

The pDOMKR1 obtained in Example (17-1) was cleaved at SacI-ClaI andtransformed into the Ogataea minuta TK8 strain (och1Δ pep4Δ prb1Δ ura3Δ)and the Ogataea minuta TK9 strain (och1Δ pep4Δ prb1Δ ura3Δ ade1Δ)obtained in Example (15-2), by electric pulse method.

The KTR1 knockout mutants were screened by subjecting the chromosomalDNAs of the obtained transformants to Southern analysis. Specifically,the chromosomal DNAs of the host strain and the transformants werecleaved with SacI and subjected to Southern analysis using the 2-kb SacIfragment isolated from the pDOMKR1 (FIG. 16) as a probe. As a result, 2kb band was detected in the host strain, while 5 kb band was detected inthe knockout mutants. After culturing the knockout mutants in the YPDmedium until stationary phase, a strain resistant to 5-fluoroorotic acid(5-FOA) was obtained. The chromosomal DNA of the 5-FOA resistant strainwas cleaved with SacI and again subjected to Southern analysis using the2-kb SacI fragment isolated from the pDOMKR1 (FIG. 16) as a probe, and astrain was selected from which the URA3 gene was deleted and for which 5kb band was detected. The och1Δ ktr1Δ pep4Δ prb1Δ ura3Δ strain obtainedwas named Ogataea minuta TK10 strain, and the och1Δ ktr1Δ pep4Δ prb1Δura3Δ ade1Δ strain was named Ogataea minuta TK11 strain.

The sensitivity of Ogataea minuta TK10 and Ogataea minuta TK11 strainsto hygromycin B was examined. Ogataea minuta IFO 10746, a wild strain,yielded colonies on a plate containing 50 μg/ml hygromycin B, butneither Ogataea minuta TK10 nor Ogataea minuta TK11 strain yielded acolony even on a plate containing 5 μg/ml hygromycin B. It is known thatsugar chain mutants of Saccharomyces cerevisiae have higher sensitivityto a drug like hygromycin B than the wild strain of the same. Thus, itwas presumed that these Ogataea minuta ktr1Δ strains had short sugarchains.

Further, in the Ogataea minuta ktr1Δ strains, the precipitation of cellswas markedly increased just like the Saccharomyces cerevisiae och1Δstrain. This may show that the sugar chains of these Ogataea minutaktr1Δ strains were short.

Example 18

Cloning of MNN9 Gene of Ogataea minuta

The MNN9 gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(18-1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to thefollowing amino acid sequences conserved in MNN9 from Saccharomycescerevisiae (GenBank accession number; L23752) and Candida albicans(GenBank accession number; U63642):

TSWVLWLDAD; (SEQ ID NO: 65) and ETEGFAKMAK (SEQ ID NO: 66)were synthesized as follows.

PMN5; 5′- (SEQ ID NO: 67) ACNWSNTGGGTNYTNTGGYTNGAYGCNGA-3′ PMN3; 5′-(SEQ ID NO: 68) TTNGCCATYTTNGCRAANCCYTCNGTYTC-3′

The primer PMN5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence TSWVLWLDAD, and the primer PMN3has a sequence complementary to the nucleotide sequence corresponding tothe amino acid sequence ETEGFAKMAK.

PCR by primers PMN5 and PMN3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 1 minute)×25 cycles). The amplified DNAfragment of approximately 0.4 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences for MNN9 genes from Saccharomycescerevisiae and Candida albicans. The 0.4-kb DNA insert was recoveredafter EcoRI cleavage of the plasmid and agarose gel electrophoresis.

(18-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (18-1) as a probe by the methoddescribed in Example (2-2). The results suggested that there existed theMNN9 gene in the BamHI fragment of approximately 8 kb. Then, to clonethe DNA fragment, a library was prepared. The chromosomal DNA of Ogataeaminuta was cleaved with BamHI and subjected to agarose gelelectrophoresis, and then the approximately 8-kb DNA fragment wasrecovered from the gel. The DNA fragment was ligated with BamHI-cleavedpUC 118 and then transformed into Escherichia coli strain DH5 α toprepare a library.

About 6,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMMN9 wasselected from the 2 positive clones obtained.

(18-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the ApaI-BglII region of the plasmid pOMMN9(FIG. 17) was determined by deletion mutant and primer walking method toobtain a nucleotide sequence represented by SEQ ID NO:69.

In the nucleotide sequence of SEQ ID NO:69, there existed an openreading frame of 1,104 bp, starting at position 931 and ends at position2,034. The homology studies between the amino acid sequence (SEQ IDNO:70) deduced from the open reading frame and the MNN9 gene productfrom Saccharomyces cerevisiae or Candida albicans showed that 59% or 62%of amino acids were respectively identical between them.

Example 19

Preparation of Ogataea minuta MNN9 Knockout Mutant

The MNN9 gene was disrupted by transformation using the URA3 gene ofOgataea minuta as a marker.

(19-1) Preparation of MNN9 Disruption Vector

As shown in FIG. 17, plasmid pDOMN9 was prepared by replacing theapproximately 1-kb SaI-BglII region of the MNN9 structural gene by theURA3 gene. To obtain a uracil auxotrophic mutant again from MNN9knockout mutants, the URA3 gene having repetitive structures before andafter the structural gene was used as a marker. The 1.2-kb ApaI-SalIfragment isolated from the plasmid pOMMN9-1 having the MNN9 gene regiondescribed in Example 18 was inserted into the ApaI-SalI of thepBluescript II SK−. The 2.2-kb NheI-BglII fragments isolated from theplasmid pOMMN9-1 and the 3.3 kb BglII-HindIII fragment isolated from thepROMU1 described in Example (8-1) were inserted into the XbaI-HindIII ofthe obtained plasmid. The resultant plasmid was named pDOMN9 (FIG. 17).

(19-2) Transformation

The pDOMN9 obtained in Example (19-1) was cleaved with ApaI andtransformed into the Ogataea minuta TK8 strain (och1Δ pep4Δ prb1Δura3Δ), the Ogataea minuta TK9 strain (och1Δ pep4Δ prb1Δ ura3Δ ade1Δ)obtained in Example (15-2) and the Ogataea minuta TK10 strain (och1Δktr1Δ pep4Δ prb1Δ ura3Δ), the Ogataea minuta TK11 strain (och1Δ ktr1Δpep4Δ prb1Δ ura3Δ ade1Δ) obtained in Example (17-2), by electric pulsemethod.

The MNN9 knockout mutants were screened by subjecting the chromosomalDNAs of the obtained transformants to Southern analysis. Specifically,the chromosomal DNAs of the host strain and the transformants werecleaved with ApaI and BglII and subjected to Southern analysis using the1.2-kb ApaI-SalI fragment isolated from the pOMMN9-1 (FIG. 17) as aprobe. As a result, a band was detected at 2.2 kb in the host strain,while a band at 5.5 kb in the knockout mutants. After culturing theknockout mutants on the YPD medium until stationary phase, a strainresistant to 5-fluoroorotic acid (5-FOA) was obtained. PCR by primersDMN5; 5′-AGATGAGGTGATTCCACGTAATTTGCCAGC-3′ (SEQ ID NO:71) and DMN3;5′-TTTTGATTGTCATCTATTTCGCACACCCTG-3′ (SEQ ID NO:72) was performed usingthe chromosomal DNA of the 5-FOA resistant strain as a template ((94° C.for 30 seconds, 60° C. for 1 minute and 72° C. for 1 minute)×25 cycles).As a result, a 1 kb amplified DNA fragment was detected in the strainfrom which the URA3 gene was deleted. The och1Δ mnn9Δ pep4Δ prb1Δ ura3Δstrain obtained was named Ogataea minuta TK12 strain, the och1Δ mnn9Δpep4Δ prb1Δ ura3Δ ade1Δ strain Ogataea minuta TK13 strain, the och1Δktr1Δ mnn9Δ pep4Δ prb1Δ ura3Δ strain was named Ogataea minuta TK14strain, and the och1Δ ktr1Δ mnn9Δ pep4Δ prb1Δ ura3Δ ade1Δ strain wasnamed Ogataea minuta TK15 strain.

The sensitivity of the Ogataea minuta TK14 and Ogataea minuta TK15strains to hygromycin B was examined. Ogataea minuta IFO 10746, a wildstrain, yielded colonies on a plate containing 50 μg/ml hygromycin B asdescribed in Example (17-2), but neither Ogataea minuta TK12 nor Ogataeaminuta TK13 strain yielded a colony even on a plate containing 20 μg/mlhygromycin B. Thus, it was presumed that these Ogataea minuta mnn9Δstrains had short sugar chains.

Example 20

Cloning of Alcohol Oxidase (AOX1) Gene of Ogataea minuta

The AOX1 gene was obtained from Ogataea minuta IFO 10746 and itsnucleotide sequence was determined.

(20-1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to thefollowing amino acid sequences conserved in alcohol oxidase from Pichiapastoris (GenBank accession number; U96967, U96968) and Candida boidinii(GenBank accession number; Q00922):

GGGSSINFMMYT; (SEQ ID NO: 73) and DMWPMVWAYK (SEQ ID NO: 74)were synthesized as follows.

(SEQ ID NO: 75) PAX5; 5′-GGNGGNGGNWSNWSNATHAAYTTYATGATGTAYAC-3′ (SEQ IDNO: 76) PAX3; 5′-TTRTANGCCCANACCATNGGCCACATRTC-3′

The primer PAX5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence GGGSSINFMMYT, and the primerPAX3 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence DMWPMVWAYK.

PCR by primers PAX5 and PAX3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 1 minute)×25 cycles). The amplified DNAfragment of approximately 1.1 kb was recovered and cloned using TOPO TACloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences for alcohol oxidase genes fromPichia pastoris and Candida boidinii. The 1.1-kb DNA insert wasrecovered after EcoRI cleavage of the plasmid and agarose gelelectrophoresis.

(20-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (20-1) as a probe by the methoddescribed in Example (2-2). The results suggested that there existedAOX1 gene in the HindIII fragment of approximately 8 kb. Then, to clonethe DNA fragment, a library was prepared. The chromosomal DNA of Ogataeaminuta was cleaved with HindIII and subjected to agarose gelelectrophoresis, and then the approximately 6-kb DNA fragment wasrecovered from the gel. The DNA fragment was ligated withHindIII-cleaved pUC 118 and then transformed into Escherichia colistrain DH5 α to prepare a library.

About 6,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMAX1 wasselected from the 6 positive clones obtained.

(20-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the HindIII-SmaI region of the plasmid pOMAX1(FIG. 18) was determined by deletion mutant and primer walking method toobtain a nucleotide sequence represented by SEQ ID NO:77.

In the nucleotide sequence of SEQ ID NO:77 there existed an open readingframe of 1,992 bp, starting at position 2,349 and ends at position4,340. The homology studies between the amino acid sequence (SEQ IDNO:78) deduced from the open reading frame and the alcohol oxidase fromPichia pastoris or Candida boidinii showed that 72% or 74% of aminoacids were respectively identical between them.

Example 21

Construction of Heterologous Gene Expression Plasmid Using AOX1 GenePromoter and Terminator

(21-1) Construction of Expression Cassette Using AOX1 Gene Promoter andTerminator

An expression cassette was constructed for transferring foreign genesbetween the Ogataea minuta AOX1 gene promoter (SEQ ID NO:79) andterminator (SEQ ID NO:80). To transfer XbaI, SmaI and BamHI sitesbetween the AOX1 gene promoter and terminator, the following primerswere synthesized:

(SEQ ID NO: 81) OAP5; 5′-CTGCAGCCCCTTCTGTTTTTCTTTTGACGG-3′ (SEQ ID NO:82) OAP3; 5′- CCCCCGGATCCAGGAACCCGGGAACAGAATCTAGATTTTTTCGTAAGTCGTAAGTCGTAACAGAACACAAGAGTCTTTGAACAAGTTGAG-3′ (SEQ ID NO: 83) OAT5; 5′-CCCCCCCGGATCCGAGACGGTGCCCGACTCTTGTTCAATTCTTTTGG-3′ (SEQ ID NO: 84) OAT3;5′-CCCATAATGGTACCGTTAGTGGTACGGGCAGTC-3′

PCR by primers OAP5 and OAP3 ((94° C. for 30 seconds, 55° C. for 1minute and 72° C. for 1 minute)×20 cycles), and PCR by primers OAT5 andOAT3 ((94° C. for 30 seconds, 55° C. for 1 minute and 72° C. for 1minute)×20 cycles) were performed using the pOMAX1 shown in FIG. 18 as atemplate. The amplified DNA fragments of 0.5 kb and 0.8 kb wererecovered and cloned using TOPO TA Cloning Kit. The nucleotide sequencesof DNA inserts were determined, and then clones having correctnucleotide sequences were selected. The DNA inserts of 0.5 kb and 0.8 kbwere isolated as PstI-BamHII fragment and BamHI-KpnI fragment,respectively. The above described 0.5-kb PstI-BamHII fragment wasinserted into the PstI-BamHII of the pOMAX1. Then, the 0.8-kb BamHI-KpnIfragment was inserted into the BamHI-KpnI of the obtained plasmid. Theresultant plasmid was named pOMAXPT1 (FIG. 18).

The pOMAXPT1 had an expression cassette controlled by the AOX1 promoterand terminator that allowed foreign genes to be transferred at the XbaI,SmaI and BamHI sites.

(21-2) Construction of Heterologous Gene Expression Plasmid Using AOX1Gene Promoter and Terminator and Using URA3 Gene as a Selectable Marker

The 3.1-kb BglIH-HindIII fragment containing the Ogataea minuta URA3gene and isolated from the pOMUR1 described in Example (5-2) wasinserted into the BamHI-HindIII of pUC19. The obtained plasmid was namedpOMUR5 (FIG. 18). The pOMUR5 was cleaved with StyI and SacI andblunt-ended, and ApaI linkers were then inserted thereinto. The obtainedplasmid was named pOMUR6. The pOMUR6 was cleaved with XbaI andblunt-ended, and ligated. The obtained plasmid was named pOMUR-X. ThepOMUR-X was cleaved with SalI and blunt-ended, and a NotI linker wasinserted thereinto.

The resultant plasmid was named pOMUR-XN. The 3.1-kb HindIII-KpnIfragment containing the expression cassette controlled by the Ogataeaminuta AOX1 promoter and terminator which was isolated from the pOMAXPT1as described in Example (21-1), was inserted into the HindIII-KpnI ofthe pOMUR-XN. The obtained plasmid was named pOMex1U (FIG. 18).

The pOMex1U was cleaved with BglII and blunt-ended, and a NotI linkerwas inserted thereinto. The obtained plasmid was named pOMex1U-NO (FIG.18). The 3.1-kb HindIII-KpnI fragment containing the expressioncontrolled by the Ogataea minuta AOX1 gene promoter and terminator whichwas isolated from the pOMex1U-NO, was inserted into the HindIII-KpnI ofthe pOMUR-X. The resultant plasmid was named pOMex2U (FIG. 18).

(21-3) Construction of Heterologous Gene Expression Plasmid Using AOX1Gene Promoter and Terminator and Using G418 Resistant Gene as aSelectable Marker

The pOMKmR1, which comprised the G418 resistant gene expression cassettecontrolled by the GAP gene promoter and terminator described in Example4, was cleaved with PstI and blunt-ended, and an ApaI linker wasinserted thereinto. The G418 resistant gene expression cassette wasisolated, as a 2.3-kb ApaI-KpnI fragment, from the obtained plasmid andinserted into the ApaI-KpnI of the POMex1U-NO described in Example(21-2). The resultant plasmid was named pOMex3G (FIG. 18).

(21-4) Construction of Heterologous Gene Expression Plasmid Using AOX1Gene Promoter and Terminator, and Using ADE1 Gene as a Selectable Marker

A plasmid was prepared by cleaving with SmaI the pOMAD1, which containedthe ADE1 gene described in Example 7, transferring an ApaI linker,cleaving with EcoRV, transferring a KpnI linker, cleaving with BglII,blunt-ending, and transferring a NotI linker. The ADE1 gene expressioncassette was isolated, as a 3.1-kb ApaI-KpnI fragment, from the obtainedplasmid, and inserted into the ApaI-KpnI containing the expressioncassette controlled by the Ogataea minuta AOX1 gene promoter andterminator which was obtained by ApaI-KpnI from the pOMex1U. Theresultant plasmid was named pOMex4A (FIG. 18).

(21-5) Construction of Heterologous Gene Expression Plasmid Using AOX1Gene Promoter and Terminator and Using Hygromycin B Resistant Gene as aSelectable Marker

To perform transformation by the selection of antibiotic hygromycin Bresistance, a plasmid containing the hygromycin B resistant gene(hygromycin B phosphotransferase gene) expression cassette wasconstructed.

To isolate the hygromycin B resistant gene, the following primers weresynthesized:

HGP5; 5′- (SEQ ID NO: 85) GTCGACATGAAAAAGCCTGAACTCACCGC-3′; and HGP3;5′-ACTAGTCTATTCCTTTGCCCTCGGACG-3′. (SEQ ID NO: 86)

PCR by primers HGP5 and HGP3 was performed using the plasmid pGARHcontaining the hygromycin B resistant gene (Applied Environ. Microbiol.,Vol. 64 (1998) p2676) as a template ((94° C. for 30 seconds, 50° C. for1 minute and 72° C. for 1 minute)×20 cycles). The 1.0 kb amplified DNAfragment was recovered and cloned using TOPO TA Cloning Kit.

The nucleotide sequence of the DNA insert was determined, and a clonehaving the correct nucleotide sequence was selected. The 1.0 kb DNAinsert was isolated as a Sal1I-EcOT22II fragment and inserted into theSalI-EcoT22I of the pOMGP4 constructed in Example 3. The obtainedplasmid was named pOMHGR1. The obtained plasmid was cleaved with HindIIIand blunt-ended, and an ApaI linker was inserted thereinto. Thehygromycin B resistant gene expression cassette was isolated, as a3.0-kb ApaI-KpnI fragment, from the obtained plasmid, and then insertedinto the ApaI-KpnI of the pOMex1U-NO described in Example 21-2. Theresultant plasmid was named pOMex5H (FIG. 18).

Example 22

Construction of Heterologous Gene Expression Plasmid Using GAP GenePromoter and Terminator, and Using URA3 Gene as a Selectable Maker

The gene expression cassette using the GAP gene promoter and terminator,as described in Example 3, was isolated as a 2.0-kb HindIII-KpnI, andthen inserted into the HindIII-KpnI of each of the pOMUR-XN described inExample (21-2) and the pOMex4A described in Example (21-4) (wherepOMex4A was a fragment comprising pUC19-ADE1). The obtained plasmidswere named pOMexGP1U and pOMexGP4A, respectively (FIG. 18).

Example 23

Construction of Aspergillus saitoi-Derived α-1,2-Mannosidase ExpressionPlasmid Ysing AOX1 Gene Promoter and Terminator

Example 11 suggested that expression of α-1,2-mannosidase in the Ogataeaminuta Δoch1 strain enabled the preparation of a Man5 producing yeast.So, Ogataea minuta Δoch1 strain in which α-1,2-mannosidase was expressedwas prepared. The Aspergillus saitoi-derived α-1,2-mannosidase gene,which comprised a signal sequence of asperginopepsin I (apnS) at theamino terminus and a yeast endoplasmic reticulum (ER) retention signal(HDEL) (SEQ ID NO: 121) at the carboxyl terminus (J. Biol. Chem., 273(1998) 26298), was used for expression. PCR by the primers:

(SEQ ID NO: 87) 5′-GGGGGGTCGACATGGTGGTCTTCAGCAAAACCGCTGCCC-3′; and (SEQID NO: 88) 5′-GGGGGGCGGCCGCGTGATGTTGAGGTTGTTGTACGGAACCCCC-3′

was performed using the plasmid pGAMH1 comprising the above describedgene as a template ((94° C. for 30 seconds, 50° C. for 1 minute and 72°C. for 30 seconds) ×20 cycles). The approximately 0.5-kb DNA fragment5′-upstream of the amplified α-1,2-mannosidase gene was recovered,cleaved with SalI and NotI, and inserted into the SalI-NotI of thepBluescript II SK-. The nucleotide sequence of the DNA insert wasdetermined and a clone comprising the correct nucleotide sequence wasselected. The 1.2-kb BglII-NotI fragment downstream of the BglII site inthe α-1,2-mannosidase gene isolated from the pGAMH1 was inserted intothe BglII-NotI of the obtained plasmid. This plasmid was named paMSN.The paMSN was cleaved with SalI and blunt-ended, and an XbaI linker wasinserted thereinto. This plasmid was named paMXN. Separately, the paMSNwas cleaved with NotI and blunt-ended, and a BamHI linker was insertedthereinto. The resultant plasmid was named paMSB. The 0.4-kb XbaI-BglIIfragment upstream of the α-1,2-mannosidase gene isolated after cleavingthe paMXN with XbaI-ApaI, and the 1.1-kb ApaI-BamHI fragment downstreamof the α-1,2-mannosidase gene isolated after cleaving the paMSB withApaI-BamHI, were inserted into the XbaI-BamHII of the pOMex1U describedin Example (21-2) and of the pOMex3G described in Example (21-3),respectively, by three points ligation. The obtained plasmids were namedpOMaM1U and pOMaM3G, respectively.

Example 24

Preparation of Aspergillus saitoi-derived α-1,2-mannosidase GeneExpressing Ogataea minuta Δoch1 Strain and Sugar Chain Analysis of Same

The pOMaM1U obtained in Example 23 was cleaved with NotI, and theOgataea minuta TK3-A strain (och1Δ ura3Δ) obtained in Example (10-2) wastransformed with it. The intracellular α-1,2-mannosidase activity of theobtained transformant was measured. The transformants cultured in theBYPM medium (0.67% yeast nitrogen base, 1% yeast extract, 2%polypeptone, 100 mM potassium phosphate buffer pH 6.0, 0.5% methanol)were harvested and suspended in 0.1 M sodium acetate buffer pH 5.0containing 1% Triton X100 and 1 mM PMSF, then the cells were disruptedwith glass beads to obtain a cell extract. The extract was appropriatelydiluted, 20 pmol of Man6b sugar chain (TAKARA SHUZO CO., LTD., Japan)was added, and the mixture was incubated for reaction at 37° C. for10-60 minutes. After the incubation, the mixture was boiled toinactivate the enzyme and subjected to HPLC to analyze the produced Man5sugar chain. The HPLC conditions were as follows.

Column: TSK-Gel ODS 80TM (6×150 mm, TOSOH CORPORATION, Japan)

Column temperature: 50° C.

Flow rate: 1.2 ml

Elution conditions: A: 100 mM ammonium acetate pH 6.0

-   -   B: 100 mM ammonium acetate pH 6.0+0.15% butanol    -   Linear gradient of 0 minute A=70% and 12 minutes A=0%

A yeast strain having the highest α-1,2-mannosidase activity wasselected and named Ogataea minuta TK3-A-MU1 strain. The yeast strain wascultured again in the BYPM medium, and the structure of the sugar chainof cell surface mannan proteins was analyzed. The preparation ofPA-oligosaccharides was carried out in accordance with the methoddescribed in Example 1. And HPLC analysis was performed by the methoddescribed in Example 11.

The results are shown in FIG. 19. The size analysis by normal phasecolumn revealed that the Ogataea minuta TK3-A-MU1 strain mainly producedMan5GlcNAc2. The structure analysis by reverse phase column revealedthat the Man5GlcNAc2 was the sugar chain of the following structuralformula 2:

which sugar chain was consistent with the human-type, high mannose-typesugar chain, and precursor of hybrid type or complex type sugar chains.

Example 25

Construction of Saccharomyces cerevisiae-derived Invertase ExpressionPlasmid Using AOX1 Gene Promoter and Terminator

Invertase (SUC2) gene of Saccharomyces cerevisiae (GenBank accessionnumber; V01311) was obtained by PCR. PCR by the primers:

(SE ID NO: 89) 5′-GGGGACTAGTATGCTTTTGCAAGCTTTCCTTTTCCTTTTG-3′; and (SEQID NO: 90) 5′-CCCCAGATCTTATTTTACTTCCCTTACTTGGAACTTGTC-3′was performed using the chromosomal DNA of Saccharomyces cerevisiaeS288C strain as a template ((94° C. for 30 seconds, 50° C. for 1 minuteand 72° C. for 1.5 minute)×20 cycles). The amplified DNA fragment ofapproximately 1.4 kb was recovered, cleaved with SpeI and BglII, andinserted into the XbaI-BamHI of the pOMex1U described in Example (21-2)and of the pOMex3G described in Example (21-3). The obtained plasmidswere named pOMIV1U and pOMIV3G, respectively.

Example 26

Transferring of Saccharomyces cerevisiae-derived Invertase Gene IntoAspergillus saitoi-derived α-1,2-mannosidase Gene Expressing Ogataeaminuta OCH1 Knockout Mutant and Expression of Same

The pOMIV3G obtained in Example 25 was cleaved with NotI and transferredinto the Ogataea minuta TK3-A-MU1 strain described in Example 24. Thetransformant was cultured in the BYPM medium (0.67% yeast nitrogen base,1% yeast extract, 2% polypeptone, 100 mM potassium phosphate buffer pH6.0, 0.5% methanol). The culture was centrifuged and the resultantsupernatant was assayed for invertase activity by the followingprocedures. Specifically, 2 μl of appropriately diluted culturesupernatant and 200 μl of 100 mM sodium acetate buffer (pH 5.0)containing 2% sucrose were mixed together and incubated at 37° C. for10-30 minutes, and 500 μl of Glucose-Test Wako (Wako Pure ChemicalIndustries, Ltd., Japan) was added to 2 μl of the reaction mixture todevelop color. An absorbance based on free glucose generated byinvertase was measured at 505 nm. The most productive yeast strainOgataea minuta TK3-A-MU-IVG1 strain produced about 600 mg invertase/lmedium, and the invertase was most part of proteins in the culturesupernatant.

Example 27

Structure Analysis of Sugar Chain of Saccharomvces cerevisiae-derivedInvertase Secreted by the Strain Prepared in Example 26

The culture supernatant of the Ogataea minuta TK3-A-MU-IVG1 strainobtained in Example 26 was concentrated by ultrafiltration using AmiconYM76 membrane (Amicon), desalted, and subjected to an anion exchangecolumn chromatography (Q-Sepharose FF, Amersham Pharmacia Biotech) topurify invertase fractions. The fractions were freeze-dried andPA-N-linked sugar chain was prepared by the method described inExample 1. The analysis by HPLC was performed by the method described inExample 11. The results are shown in FIG. 20. The results of the sizeanalysis by amide column revealed that 90% or more sugar chains of theinvertase was composed of Man5GlcNAc2. The structure analysis by reversephase column showed that the Man5GlcNAc2 was the sugar chain representedby the structural formula 2 described in Example 24:

This sugar chain was consistent with the Man5 type, high mannose typesugar chain, which is a precursor of hybrid type or complex type sugarchain.

Example 28

Preparation of Human Antibody Gene-transferred Ogataea minuta OCH1Knockout Mutant, Transfer and Expression of Aspergillus saitoi-derivedα-1,2-mannosidase Gene in the Mutant, and Production of Human AntibodyUsing Same

Anti-human G-CSF antibody gene was transferred into the Ogataea minutaTK9 strain (och1Δ pep4Δ prb1Δ ura3Δ ade1Δ) obtained in Example (15-2).

Anti-human G-CSF antibody producing hybridoma was obtained by producinga mouse producing anti-human G-CSF antibodies using human G-CSF as anantigen in accordance with the method by Tomiduka et al. (Proc. Natl.Acad. Sci. U.S.A. 97(2), 722-7 (2000)), removing the spleen from themouse by conventional procedure (Muramatsu et al., Jikken SeibutsugakuKoza, Vol. 14, pp. 348-364 ), and fusing the B cells with a mousemyeloma. The antibody gene was obtained from the hybridoma by the methoddescribed by Welschof, M et al. (J. Immunol. Methods. 179 (2), 203 -14(1995)).

XbaI linker and BamHI linker were added at the N-terminus and theC-terminus, respectively, of each of the anti-G-CSF light chain gene(SEQ ID NO:91; the coded amino acid sequence, SEQ ID NO:92) andanti-G-CSF heavy chain gene (SEQ ID NO:93, the coded amino acidsequence, SEQ ID NO:94). Subsequently, the light chain gene wastransferred at the XbaI-BamHI site of the pOMex4A described in Example(21-4) while the heavy chain gene at the XbaI-BamHI site of the pOMex3Gdescribed in Example (21-3), respectively. Each of the constructedexpression vectors was cleaved with NotI, and the Ogataea minuta TK9strain was in turn transformed. The obtained transformants were culturedin the BYPMG medium (0.67% yeast nitrogen base, 1% yeast extract, 2%polypeptone, 100 mM potassium phosphate buffer pH 6.0, 0.1% methanol,0.2% glycerol) at 20° C. for 72 hours, and then centrifuged. The culturesupernatant was subjected to Western analysis using a horseradishperoxidase labeled anti-human IgG sheep antibody (Amersham PharmaciaBiotech). First, 100 μl of the culture supernatant was concentratedthrough Microcon YM30 membrane and subjected to SDS-PAGE. Then, theelectrophoresed proteins were blotted on PVDF membrane (Immobilon,Millipore), which membrane was then blocked over 1 hour using Block Ace(Dainippon Pharmaceutical Co., Ltd., Japan). Proteins on the membranewere incubated for 1 hour in TBS solution (Tris buffer containing 0.15 MNaCl) containing the horseradish peroxidase labeled anti-human IgG sheepantibody (1000:1 dilution), and unbound antibodies were washed out withTBS containing 0.04% Tween 20. The detection of signal was carried outusing Super Signal WestDura (Pierce). Thus, the transformant producingthe antibody in the culture supernatant was selected. The Ogataea minutaTK9-derived antibody producing strain was named Ogataea minuta TK9-IgB1.

Then, the Aspergillus saitoi-derived α-1,2-mannosidase gene wastransferred into the Ogataea minuta TK9-IgB1 strain. Aftertransformation, α-1,2-mannosidase expressing strain was selected fromthe obtained transformants by the method described in Example 24 usingthe plasmid pOMaM1U prepared in Example 23. The resultant strain wasnamed Ogataea minuta TK9-IgB-aM. This strain was cultured in the BYPMGmedium at 20° C. for 72 hours and centrifuged. The culture supernatantobtained by the centrifugation was subjected to Western analysis.

The results are shown in FIG. 21. The results revealed that the Ogataeaminuta TK9-IgB-aM strain produced both antibody heavy chains and lightchains, although part of the antibody heavy chains was degraded.

Further, the culture supernatant of the Ogataea minuta TK9-IgB-aM strainwas concentrated by ultrafiltration using Amicon YM76 membrane (Amicon),desalted, and subjected to Protein A column chromatography (Hi-TrapProteinA HP, Amersham Pharmacia Biotech) to purify the antibodyfractions through the elution with glycine-HCl, pH 3.0 (FIG. 22). Todetect the binding of the antibody to G-CSF as the antigen, Westernanalysis was performed. The analysis was done in accordance with theabove described procedures using the purified antibody as a primaryantibody and the horseradish peroxidase labeled anti-human IgG sheepantibody as a secondary antibody. The results are shown in FIG. 23. Theresults revealed that the antibody produced by the Ogataea minutaTK9-IgB1 strain bound to G-CSF as the antigen.

Example 29

Structure Analysis of Sugar Chains of Human Antibody Produced by theStrains Prepared in Example 28

The purified antibodies produced using the Ogataea minuta TK9-IgB-aMstrain and the Ogataea minuta TK9-IgB strain as shown in Example 28 weredialyzed and freeze-dried. PA-N-linked sugar chains were prepared by themethod described in Example 11 and subjected to size analysis by normalphase column. The results are shown in FIG. 24. The results revealedthat the sugar chain of the antibody produced by the Ogataea minutaTK9-IgB strain was composed mainly of Man₇GlcNAc₂, while the sugar chainof the antibody produced by the Ogataea minuta TK9-IgB-aM strain wascomposed mainly of Man₅GlcNAc₂, which was a mammalian type, high mannosetype sugar chain. The results indicated that 80% or more sugar chainswere composed of Man₅GlcNAc₂.

Example 30

Cloning of HIS3 (Imidazoleglycerol Phosphate Dehydratase) Gene fromOgataea minuta

The HIS3 gene was obtained from Ogataea minuta IFO 10746 strain, and itsnucleotide sequence was determined.

(30- 1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to the aminoacid sequences conserved in HIS3 gene products from Saccharomycescerevisiae (Accession number; CAA27003) and Pichia pastoris (Accessionnumber; Q92447):

VGFLDHM; (SEQ ID NO: 95) and PSTKGVL (SEQ ID NO: 96)were synthesized as follows.

PHI5; 5′-TNGGNTTYYTNGAYCAYATG-3′ (SEQ ID NO: 97) PHI3;5′-ARNACNCCYTTNGTNSWNGG-3′ (SEQ ID NO: 98)

The primer PHI5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence VGFLDHM, and the primer PHI3has a sequence complementary to the nucleotide sequence corresponding tothe amino acid sequence PSTKGVL.

PCR by primers PHI5 and PHI3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 strain as a template ((94° C. for 30 seconds,50° C. for 30 seconds and 72° C. for 1 minute)×25 cycles). The amplifiedDNA fragment of approximately 0.5 kb was recovered and cloned using TOPOTA Cloning Kit. Plasmid DNA was isolated from the obtained clone andsequenced. For a DNA insert of the plasmid, a clone was selected whichhad a nucleotide sequence encoding an amino acid sequence highlyhomologous to the amino acid sequences of HIS3 gene products fromSaccharomyces cerevisiae and Pichia pastoris. The 0.5-kb DNA insert wasrecovered after EcoRI digestion of the plasmid and agarose gelelectrophoresis.

(30-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (30-1) as a probe by the methoddescribed in Example (2-2). The results indicated that there existed theHIS3 gene in the PstI fragment of approximately 4 kb. Then, to clone theDNA fragment, a library was constructed. The chromosomal DNA of Ogataeaminuta was cleaved with PstI and subjected to agarose gelelectrophoresis, and then the approximately 4-kb DNA fragment wasrecovered from the gel. The recovered DNA fragment was ligated withPstI-cleaved and BAP-treated pUC 118 and then transformed intoEscherichia coli DH5 α strains to prepare a library.

About 2,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMHI1 wasselected from the 4 positive clones obtained.

(30-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the PstI-PstI region of the plasmid pOMHI1(FIG. 25) was determined by primer walking method to obtain a nucleotidesequence represented by SEQ ID NO:99.

In the nucleotide sequence of SEQ ID O: 99, there existed an openreading frame of 714 bp, starting at position 1,839 and ends at position2,552. The homology studies between the amino acid sequence (SEQ ID NO:100) deduced from the open reading frame and the HIS3 gene product fromSaccharomyces cerevisiae or Pichia pastoris showed that 73% or 71% ofamino acids were respectively identical between them.

Example 31

Preparation of Ogataea minuta HIS3 Knockout Mutant

The HIS3 gene was disrupted by transformation using the Ogataea minutaURA3 gene as a marker.

(31-1) Preparation of HIS3 Gene Disruption Vector

As shown in FIG. 25, plasmid pDOMH1 was prepared by replacing theapproximately 70 bp region of the HIS3 structural gene by the URA3 gene.

The plasmid pROMU1 described in Example 8-1 was cleaved with BglII,blunt-ended, and ligated with an EcoT22I linker. The obtained plasmidwas named pROMUHT.

The plasmid pOMHI1 containing the HIS3 gene region and described inExample (30-3) was cleaved with PflMI, blunt-ended, and ligated with anEcoT22I linker. The obtained plasmid was named pOMHI2. This plasmid wasthen cleaved with EcoRI and SalI and ligated with the EcoRI- andSalI-cleaved pBluescript II KS+. The obtained plasmid was named pOMHI3.The pOMHI3 was cleaved with BtgI, blunt-ended, and ligated with aHindIII linker. The obtained plasmid was named pOMHI4. The 3.3-kbEcoT22I-HindIII fragment isolated from the pROMUHT was inserted into theEcoT22I-HindIII of the obtained plasmid. The resultant plasmid was namedpDOMHI1 (FIG. 25).

(31-2) Transformation

The pDOMHI1 obtained in Example (30-2) was cleaved with BamHI and XhoIand transformed into the Ogataea minuta TK11 strain (och1Δ ktr1Δ pep4Δprb1Δ ura3Δ ade1Δ) obtained in Example (17-2) by electric pulse method.To confirm that the HIS3 gene was disrupted, the following primers weresynthesized (see FIG. 26 with regard to the position of each primer):

DHI5; 5′-GGCCCAATAGTAGATATCCC-3′ (SEQ ID NO: 101) DHI3;5′-CACGGCCCGTGTAGCTCGTGG-3′ (SEQ ID NO: 102)

PCR by primers DHI5 and DHI3 was performed using the chromosomal DNAisolated from the transformant as a template ((94° C. for 30 seconds,60° C. for 1 minute and 72° C. for 2 minutes)×25 cycles). As shown inFIG. 26, a 4.6 kb amplified DNA fragment was detected in the strainwhose HIS3 locus had the plasmid integrated thereinto. The selectedstrain was cultured on the YPD medium until stationary phase and astrain resistant to 5-fluoroorotic acid (5-FOA) was obtained. PCR byprimers DHI5 and DHI3 was performed using the chromosomal DNA of the5-FOA resistant strain as a template ((94° C. for 30 seconds, 60° C. for1 minute and 72° C. for 3 minutes)×25 cycles). As shown in FIG. 26, inthe strain from which the URA3 gene was deleted, a 2 kb amplified DNAfragment was detected. This och1Δ ktr1Δ pep4Δ prb1Δ ura3Δ ade1Δ his3Δstrain was named Ogataea minuta YK1.

Example 32

Construction of Heterologous Gene Expression Plasmid Using AOX1 GenePromoter and Terminator and HIS3 Gene as a Selectable Marker

A plasmid was prepared by the steps of cleaving with SacI the pOMHI1containing the HIS3 gene as described in Example (30-3); blunt-ending;transferring an ApaI; cleaving with NcoI; blunt-ending; transferring aKpnI linker; cleaving with EcoRI; blunt-ending; and transferring a NotIlinker. The HIS3 gene expression cassette was isolated, as a 2.6-kbApaI-KpnI fragment, from the obtained plasmid, and inserted into theApaI-KpnI of the POMex1U. The resultant plasmid was named pOMex6HS (FIG.32).

The approximately 1.4-kb SpeI-BglII fragment comprising Saccharomycescerevisiae-derived invertase gene, which was prepared in Example 25, wasinserted into the XbaI-BamHI of the pOMex6HS to prepare pOMIV6HS. Thisplasmid was cleaved with NotI and transferred into the Ogataea minutaYK1 strain described in Example (31-2). The transformants were culturedin the BYPM medium (0.67% yeast nitrogen base, 1% yeast extract, 2%polypeptone, 100 mM potassium phosphate buffer pH 6.0, 0.5% methanol).The culture was centrifuged, and invertase activity was measured for thesupernatant by the following procedures. Specifically, 2 μl of theculture supernatant appropriately diluted and 200 μl of 100 mM sodiumacetate buffer (pH 5.0) containing 2% sucrose were mixed and incubatedat 37° C. for 10-30 minutes, and then 500 μl of Glucose-Test Wako (WakoPure Chemical Industries, Ltd., Japan) was added to the reaction mixtureto develop color. An absorbance based on free glucose generated byinvertase was measured at 505 nm. In the yeast strain Ogataea minutaYK1-WH1, a significant amount of invertase was produced in the medium.

Example 33

Cloning of LEU2 (3-isopropylmalate dehydrogenase) Gene from Ogataeaminuta

The LEU2 gene was obtained from Ogataea minuta strain IFO 10746, and itsnucleotide sequence was determined.

(33-1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to the aminoacid sequences conserved in LEU2 gene products from Saccharomycescerevisiae (Accession number; CAA27459) and Pichia angusta (P34733):

AVGGPKWG; (SEQ ID NO: 103) and AAMMLKL (SEQ ID NO: 104)were synthesized as follows.

PLE5; 5′- (SEQ ID NO: 105) GCNGTNGGNGGNCCNAARTGGGG-3′ PLE3;5′-NARYTTNARCATCATNGCNGC-3′ (SEQ ID NO: 106)

The primer PLE5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence AVGGPKWG, and the primer PLE3has a sequence complementary to the nucleotide sequence corresponding tothe amino acid sequence AAMMLKL.

PCR by primers PLE5 and PLE3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. recovered and cloned using TOPO TA Cloning Kit.Plasmid DNA was isolated from the obtained clone and sequenced. For aDNA insert of the plasmid, a clone was selected which had a nucleotidesequence encoding an amino acid sequence highly homologous to the aminoacid sequence of LEU2 gene products from Saccharomyces cerevisiae andPichia angusta. The 0.7-kb DNA insert was recovered after EcoRI cleavageof the plasmid and agarose gel electrophoresis.

(33-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 strain was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (33-1) as a probe by the methoddescribed in Example (2-2). The results suggested that there existed theLEU2 gene in the BamHI-ClaI fragment of approximately 6 kb. Then, toclone the DNA fragment, a library was prepared. The chromosomal DNA ofOgataea minuta was cleaved with BamHI and ClaI and subjected to agarosegel electrophoresis, and then the approximately 6-kb DNA fragment wasrecovered from the gel. The recovered DNA fragment was ligated withBamHI- and ClaI-cleaved pBluescript II KS+ and then transformed intoEscherichia coli strain DH5 α to prepare a library.

About 3,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMYP1 wasselected from the 7 positive clones obtained.

(33-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the BamHI-ClaI region of the plasmid pOMLE1(FIG. 28) was determined by primer walking method to obtain thenucleotide sequence represented by SEQ ID NO:107.

In the nucleotide sequence of SEQ ID NO:107, there existed an openreading frame of 1,089 bp, starting at position 1,606 and ends atposition 2,694. The homology studies between the amino acid sequence(SEQ ID NO:108) deduced from the open reading frame and the LEU2 geneproduct from Saccharomyces cerevisiae or Pichia angusta showed that 80%or 85% of amino acids were respectively identical between them.

Example 34

Preparation of Ogataea minuta LEU2 Knockout Mutant

The LEU2 gene was disrupted by transformation using the URA3 gene ofOgataea minuta as a marker.

(34-1) Preparation of LEU2 Gene Disruption Vector

As shown in FIG. 28, plasmid pDOMLE1 was prepared by replacing theapproximately 540-bp region of the LEU2 structural gene by the URA3gene. To obtain a uracil auxotrophic mutant again from LEU2 geneknockout mutants, the URA3 gene having repetitive structures before andafter the structural gene was used as a marker. The pROMUHT described inExample (31-1) was cleaved with HindIII, blunt-ended, and ligated with aNheI linker. The obtained plasmid was named pROMUNT.

The pOMLE1 was cleaved with StuI, blunt-ended, and ligated with a NheIlinker. The obtained plasmid was named pOMLE2. The 3.3-kb Nhe-EcoT22Ifragment isolated from the pOMURNT was inserted into the NheI-PstI ofthe pOMLE2. The obtained plasmid was named pDOMLE1.

(34-2) Transformation

The pDOMLE1 obtained in Example (34-1) was cleaved with BamHI and ClaI,and transformed into the Ogataea minuta TK11 strain (och1Δ ktr1Δ pep4Δprb1Δ ura3Δ ade1Δ) obtained in Example (17-2) by electric pulse method.To confirm that the LEU2 gene of these strains was disrupted, thefollowing primers were synthesized (see FIG. 29 with regard to theposition of each primer):

DL5; 5′-CAGGAGCTACAGAGTCATCG-3′ (SEQ ID NO: 109) DL3;5′-ACGAGGGACAGGTTGCTCGC-3′ (SEQ ID NO: 110)

PCR by primers DL5 and DL3 was performed using the chromosomal DNAisolated from the transformant as a template ((94° C. for 30 seconds,60° C. for 1 minute and 72° C. for 2 minutes)×25 cycles). As shown inFIG. 29, a 4 kb amplified fragment was detected in the strain whose LEU2locus had the plasmid integrated thereinto. The selected strain wascultured on the YPD medium until stationary phase, and a strainresistant to 5-fluoroorotic acid (5-FOA) was obtained. PCR by primersDL5 and DL3 was performed using the chromosomal DNA of the 5-FOAresistant strain as a template ((94° C. for 30 seconds, 60° C. for 1minute and 72° C. for 3 minutes)×25 cycles). As shown in FIG. 29, in thestrain from which the URA3 gene was deleted, a 1.6 kb amplified DNAfragment was detected. This och1Δ ktr1Δ pep4Δ prb1Δ ura3Δ ade1Δ leu2Δstrain was named Ogataea minuta YK2.

Example 35

Construction of Heterologous Gene Expression Plasmid Using AOX1 GenePromoter and Terminator, and LEU2 Gene as a Selectable Marker

The pOMLE1 comprising the LEU2 gene described in Example (33-2) wascleaved with PmaCI, ligated with an ApaI linker, cleaved with BamHI,blunt-ended, and ligated with a KpnI linker. The LEU2 gene expressioncassette was isolated, as a 3.3-kb ApaI-KpnI fragment, from the obtainedplasmid, and then inserted into the ApaI-KpnI of the POMex1U. Theobtained plasmid was cleaved with SpeI, blunt-ended, and ligated with aNotI linker. The resultant plasmid was named pOMex7L (FIG. 30).

The approximately 1.4-kb SpeI-BglII fragment comprising theSaccharomyces cerevisiae-derived invertase gene, obtained in Example 25,was inserted into the XbaI-BamHI of the pOMex7L to prepare pOMIV7L. Thisplasmid was cleaved with NotI and transferred into the Ogataea minutaYK2 strain described in Example (34-2). The transformant was cultured inthe BYPM medium (0.67% yeast nitrogen base, 1% yeast extract, 2%polypeptone, 100 mM potassium phosphate buffer pH 6.0, 0.5% methanol).The culture was centrifuged and the supernatant was measured forinvertase activity by the following procedures. Specifically, 2 μl ofthe culture supernatant appropriately diluted and 200 μl of 100 mMsodium acetate buffer (pH 5.0) containing 2% sucrose were mixed togetherand incubated at 37° C. for 10-30 minutes, and 500 μl of Glucose-TestWako (Wako Pure Chemical Industries, Inc., Japan) was added to the 2 μlof the reaction mixture to develop color. The absorbance based on freeglucose generated by invertase was measured at 505 nm. In the mostproductive yeast strain Ogataea minuta YK2-IVL1, a significant amount ofinvertase was produced in the medium.

Example 36

Cloning of YPS1 Gene from Ogataea minuta

The YPS1 gene was obtained from Ogataea minuta IFO 10746, and itsnucleotide sequence was determined.

(36-1) Preparation of Probe

Oligonucleotides having nucleotide sequences corresponding to thefollowing amino acid sequences conserved in YPS1 gene products fromSaccharomyces cerevisiae (Accession number; NP_(—)013221) and Candidaalbicans (Accession number; AAF66711):

DTGSSDLW; (SEQ ID NO: 111) and FGAIDHAK (SEQ ID NO: 112)were synthesized as follows.

PLE5; 5′-GAYACNGGHTCNTCNGAYYTNTGG-3′ (SEQ ID NO: 113) PLE3;5′-TTYGGHGCNATYGAYCAYGCNAA-3′ (SEQ ID NO: 114)

The primer PYP5 has a sequence complementary to the nucleotide sequencecorresponding to the amino acid sequence DTGSSDLW, and the primer PYP3has a sequence complementary to the nucleotide sequence corresponding tothe amino acid sequence FGAIDHAK.

PCR by primers PYP5 and PYP3 was performed using the chromosomal DNA ofOgataea minuta IFO 10746 as a template ((94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 1 minute)×25 cycles). Approximately 0.6 kbamplified DNA fragment was recovered and cloned using TOPO TA CloningKit. Plasmid DNA was isolated from the obtained clone and sequenced. Fora DNA insert of the plasmid, a clone was selected which had a nucleotidesequence encoding an amino acid sequence highly homologous to the aminoacid sequences of YPS1 gene products from Saccharomyces cerevisiae andCandida albicans. The 0.6-kb DNA insert was recovered after EcoRIdigestion of the plasmid and agarose gel electrophoresis.

(36-2) Preparation of Library and Screening

The chromosomal DNA of Ogataea minuta IFO 10746 was cleaved withdifferent restriction enzymes, and subjected to Southern analysis usingthe DNA fragment obtained in Example (36-1) as a probe by the methoddescribed in Example (2-2). The results suggested that there existedYPS1 gene in the EcoRI fragment of approximately 4 kb. Then, to clonethe DNA fragment, a library was constructed. The chromosomal DNA of theOgataea minuta was cleaved with EcoRI and subjected to agarose gelelectrophoresis, and then the approximately 6-kb DNA fragment wasrecovered from the gel. The recovered DNA fragment was ligated withEcoRI-cleaved and BAP-treated pUC118 and then transformed intoEscherichia coli strain DH5 α to prepare a library.

About 2,000 clones were screened by colony hybridization using the abovedescribed DNA fragment as a probe. A clone bearing plasmid pOMYP1 wasselected from the 4 positive clones obtained.

(36-3) Sequencing of Nucleotide Sequence

The nucleotide sequence of the EcoRI region of the plasmid pOMLE1 (FIG.31) was determined by primer walking method to obtain a nucleotidesequence represented by SEQ ID NO:115.

In the nucleotide sequence of SEQ ID NO:115, there existed an openreading frame of 1,812 bp, starting at 1position 1,712 and ends atposition 3,523. The homology studies between the amino acid sequence(SEQ ID NO:16) deduced from the open reading frame and the YPS1 geneproduct from Saccharomyces cerevisiae or Candida albicans showed that40% or 27% of amino acids were respectively identical between them.

Example 37

Preparation of Ogataea minuta YPS1 Knockout Mutant

The YPS1 gene was disrupted by transformation using the UR43 gene ofOgataea minuta as a marker.

(37- 1) Preparation of YPS1 Gene Disruption Vector

As shown in FIG. 31, plasmid pDOMYP1 was prepared by replacing theapproximately 300-bp region of the YPS1 structural gene by the URA3gene. To obtain a uracil auxotrophic mutant again from YPS1 knockoutmutants, the URA3 gene having repetitive structures before and after thestructural gene was used as a marker. The pROMUHT described in Example(31-1) was cleaved with HindIII, blunt-ended, and ligated with anEcoT22I linker. The obtained plasmid was named pROMUTT.

The pOMYP1 was cleaved with EcoRI, and the obtained fragment was ligatedwith EcoRI-cleaved and BAP-treated pBluescript II KS+. The obtainedplasmid was named pOMYP2. This plasmid was cleaved with BsiWI andblunt-ended, and an EcoT22I linker was inserted thereinto. The obtainedplasmid was named pOMYP3. The 3.3-kb EcoT22I fragment isolated from thepOMURTT was inserted at the EcoT22I of the pOMYP3. The obtained plasmidwas named pDOMYP1.

(37-2) Transformation

The pDOMYP1 obtained in Example (37-1) was cleaved with BamHI and ClaI,and transformed into the Ogataea minuta TK11 strain (och1Δ ktr1Δ pep4Δprb1Δ ura3Δ ade1Δ) obtained in Example (17-2) by electric pulse method.To confirm that the YPS1 gene was disrupted, the following primers weresynthesized (see FIG. 32 with regard to the position of each primer).

DY5; 5′-CTCAAGGGCCTGGAGACTACG-3′ (SEQ ID NO: 117) DY3;5′-CGGGATTCCCGAGTCGCTCACC-3′ (SEQ ID NO: 118)

PCR by primers DY5 and DY3 was performed using the chromosomal DNAisolated from the transformant as a template ((94° C. for 30 seconds,60° C. for 1 minute and 72° C. for 2 minutes)×25 cycles). As shown inFIG. 8, a 3.7 kb amplified DNA fragment was detected in the strain whoseYPS1 locus had the plasmid integrated thereinto. The selected strain wascultured on the YPD medium until stationary phase, and a strainresistant to 5-fluoroorotic acid (5-FOA) was obtained. PCR by primersDY5 and DY3 was performed using the chromosomal DNA of the 5-FOAresistant strain as a template ((94° C. for 30 seconds, 60° C. for 1minute and 72° C. for 3 minutes)×25 cycles). As shown in FIG. 32, a 1.2kb amplified DNA fragment was detected in the strain from which the URA3gene was deleted. This och1Δ ktr1Δ pep4Δ prb1Δ ura3Δ ade1Δ yps1Δ strainwas named Ogataea minuta YK3.

Example 38

Transferring of Human Antibody Gene into Ogataea minuta YPS1 KnockoutMutant and Expression of Same

Human G-CSF light chain gene (SEQ ID NO:91) and heavy chain gene (SEQ IDNO:92) were transferred into the Ogataea minuta YK3 strain (och1Δ ktr1Δpep4Δ prb1Δ ura3Δ ade1Δ yps1Δ) obtained in Example (37-2). The plasmidvector expressing anti-G-CSF light chain and heavy chain genes,described in Example 28, was cleaved with NotI, the Ogataea minuta YK3strain was transformed in turn. In accordance with the method describedin Example 28, a transformant that produced the antibodies in theculture supernatant was selected from the obtained transformants, andthe Ogataea minuta YK3-derived antibody producing strain was namedOgataea minuta YK3-IgB1.

Then Aspergillus saitoi-derived α-1,2-mannosidase gene was transferredinto the Ogataea minuta YK3-IgB1 strain. After transformation using theplasmid pOMaM1U prepared in Example 23 by the method described inExample 24, an α-1,2-mannosidase expressing strain was selected from theobtained transformants. The resultant strain was named Ogataea minutaYK3-IgB-aM. The Ogataea minuta YK3-IgB-aM strain and the Ogataea minutaTK9-IgB-aM strain prepared in Example 28 as a control were cultured inthe BYPMG medium at 28° C. for 72 hours and centrifuged. The culturesupernatant obtained by the centrifugation was subjected to Westernanalysis. The results are shown in FIG. 33. The results revealed that inantibody molecules produced by the Ogataea minuta TK9-IgB-aM strain, asa control, molecules with degraded heavy chains were detected, whereasin the antibody molecules produced by the Ogataea minuta YK3-IgB-aMstrain, the degradation of the heavy chains was retarded.

Further, the culture supernatant of the Ogataea minuta YK3-IgB-aM strainwas concentrated by ultrafiltration using an Amicon YM76 membrane(Amicon), desalted, and subjected to Protein A column chromatography(Hi-Trap ProteinA HP, Amersham Pharmacia Biotech) to purify the antibodyfractions through the elution with glycine—HCl, pH 3.0. Western analysiswas performed for the purified antibody samples (FIG. 34). The resultsof SDS-PAGE under non-reducing conditions, it was found that afull-length antibody molecule, which was composed mainly of two lightchain molecules and two heavy chain molecules, was produced. The bindingof the purified antibody to G-CSF was confirmed by the method describedin Example 28. The antibody was dialyzed and freeze-dried. PA-N-linkedsugar chains were prepared by the method described in Example 11 andsubjected to size analysis by normal phase column. From the results, itwas confirmed that the sugar chain of the antibody containedMan₅GlcNAc₂, which was a mammalian and high mannose type sugar chain.

Example 39

Transferring of a Molecular Chaperone Protein Disulfide Isomerase (PDD)Gene into Human Antibody Producing Strain Prepared in Example 38, andExpression of Same

The results obtained above confirmed that the Ogataea minuta YK3-IgB1-aMstrain produced only a trace amount of the antibody in the culturesupernatant, while the results of the Western analysis revealed that asignificant amount of the antibody was accumulated in the cells (FIG.35, lanes 1, 5). As it was presumed that the antibody protein was notfully folded, we attempted to express Protein Disulfide Isomerase (PDI)gene, as a molecular chaperone. To express the PDI gene, we constructeda plasmid, which expressed PDI gene using AOX1 gene promoter and ahygromycin resistant gene as a selectable marker.

To obtain the PDI gene (M62815) from Saccharomyces cerevisiae, thefollowing primers corresponding to the N-and C-termini of the PDI weresynthesized.

PDI5; 5′- (SEQ ID NO: 119) TCTAGAATGAAGTTTTCTGCTGGTGCCGTCCTG- 3′ PDI3;5′- (SEQ ID NO: 120) GGATCCTTACAATTCATCGTGAATGOCATCTTC- 3′

PCR by primers PDI5 and PDI3 was performed using the chromosomal DNA ofSaccharomyces cerevisiae S288C as a template ((94° C. for 30 seconds,55° C. for 1 minute and 72° C. for 1 minute)×20 cycles). 1.5 kbamplified DNA fragment was recovered and cloned using TOPO TA CloningKit. The nucleotide sequence of the DNA insert was determined and aclone having the correct nucleotide sequence was selected. The PDI geneof Saccharomyces cerevisiae can be isolated as a SpeI-BamHI fragment.

Then, the XbaI-BamHI fragment comprising the above-described PDI genewas inserted into the XbaI-BamHI of the expression cassette using theOgataea minuta AOX1 gene promoter and terminator, as prepared in Example(21-5), and the expression plasmid pOMex5H comprising the hygromycinresistant gene as a selectable marker. The resultant plasmid was namedpOMex5H-PDI.

The pOMex5H-PDI was cleaved with NotI, and the Ogataea minutaYK3-IgB1-aM strain was transformed therewith. The transformants werecultured in the BYPMG medium and centrifuged, the culture supernatantobtained by the centrifugation was subjected to Western analysis in thesame manner as in Example 38, and a transformant that produced theantibody in the culture supernatant was selected. The Ogataea minutaYK3-IgB-aM-derived antibody producing strain was named Ogataea minutaYK3-IgB-aM-P. The Ogataea minuta YK3-IgB-aM-P strain produced asignificant amount of the full-length antibody molecule as compared withthe original strain Ogataea minuta YK3-IgB-aM into which no molecularchaperon was transferred (FIG. 35, lane 4), and in which the amount ofantibody accumulated in the cells was decreased (FIG. 35, lane 6).

The antibody fractions were purified from the culture supernatant of theOgataea minuta YK3-IgB-aM strain by the method described in Example 38.The antibody fractions were dialyzed and freeze-dried. PA-N-linked sugarchains were prepared by the method described in Example 11, andsubjected to size analysis by normal phase column to confirm that thesugar chain of the antibody produced by the Ogataea minuta YK3-IgB-aMstrain contained Man₅GlcNAc₂, which was a mammalian type, high mannosetype sugar chain.

INDUSTRIAL APPLICABILITY

Using the methylotrophic yeast carrying a sugar chain mutation, which isnewly prepared by genetic engineering techniques of the invention, aneutral sugar chain identical with a high mannose type sugar chainproduced by mammalian cells such as human cells, or a glycoproteinhaving the same neutral sugar chain, can be produced in a large amountat a high purity. Further, by transferring a mammalian type sugar chainbiosynthesis-associated gene(s) into the above described mutant strain,a hybrid type or complex type mammalian sugar chain or a proteincomprising mammalian type sugar chain can be efficiently produced. Theyeast strains and glycoproteins of the invention are applicable tomedicaments, etc.

The disclosure of all the publications, patents and patent applicationscited herein is incorporated herein by reference.

1. A process for producing a methylotrophic yeast that produces amammalian type sugar chain, which comprises the steps of: 1) disruptingan OCH1 gene which encodes α-1,6-mannosyl transferase and YPS1 genewhich encodes Aspartic protease 3, in a methylotrophic yeast; and 2)introducing an α-1,2-mannosidase gene into the yeast and expressing ittherein, wherein the methylotrophic yeast belongs to the genus Pichia orOgataea.
 2. A process according to claim 1, wherein the mammalian typesugar chain is represented by the following structural formula(Man₅GlcNAc₂):


3. A process according to claim 1 or 2, wherein the methylotrophic yeastis Ogataea minuta.
 4. A process according to claim 1, wherein themethylotrophic yeast is a strain from Ogataea minuta strain IFO 10746.5. A process according to claim 1, wherein the α-1,2-mannosidase gene isexpressed under the control of a methanol-inducible promoter.
 6. Aprocess according to claim 5, wherein the methanol-inducible promoter isa promoter of an alcohol oxidase (AOX) gene.
 7. A process according toclaim 6, wherein the alcohol oxidase (AOX) gene is from Ogataea minuta.8. A process according to claim 1, characterized in that theα-1,2-mannosidase expressed from the α-1,2-mannosidase gene furthercomprises a yeast endoplasmic reticulum (ER) retention signal.
 9. Aprocess according to claim 1, wherein the α-1,2-mannosidase gene is fromAspergillus saitoi.
 10. A process according to claim 1, which furthercomprises a step of transforming a heterologous gene into the yeast. 11.A process according to claim 10, wherein the heterologous gene istransferred using an expression vector and is expressed in the yeast.12. A process according to claim 11, wherein the expression vectorcomprises a methanol-inducible promoter.
 13. A process according toclaim 12, wherein the methanol-inducible promoter is a promoter of analcohol oxidase (AOX) gene.
 14. A process according to claim 13, whereinthe alcohol oxidase (AOX) gene is from Ogataea minuta.
 15. A processaccording to claim 11, wherein the expression vector comprises apromoter of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. 16.A process according to any one of claims 10 to 15, wherein 20% or moreof N-linked sugar chains on the protein encoded by the heterologous geneis the mammalian type sugar chain represented by Structural Formula 2.17. A process according to any one of claims 10 to 15, wherein 40% ormore of N-linked sugar chains on the protein encoded by the heterologousgene is the mammalian type sugar chain represented by Structural Formula2.
 18. A process according to any one of claims 10 to 15, wherein 60% ormore of N-linked sugar chains on the protein encoded by the heterologousgene is the mammalian type sugar chain represented by Structural Formula2.
 19. A process according to any one of claims 10 to 15, wherein 80% ormore of N-linked sugar chains on the protein encoded by the heterologousgene is the mammalian type sugar chain represented by Structural Formula2.
 20. A process according to any one of claims 10 to 15, wherein theprotein encoded by the heterologous gene is from humans.
 21. A processaccording to any one of claims 10 to 15, wherein the protein encoded bythe heterologous gene is an antibody or a fragment thereof.
 22. Amethylotrophic yeast produced by a process according to claim 1 or claim10.
 23. A process for producing a protein encoded by a heterologousgene, wherein the process comprises culturing the methylotrophic yeastproduced by a process according to claim 10 in a medium to obtain theprotein encoded by the heterologous gene comprising a mammalian typesugar chain from the culture.
 24. A process for producing an Ogataeaminuta strain, which produces a mammalian type sugar chain representedby the following structural formula (Man₅GlcNAc₂):

comprising a step of disrupting OCH1 gene in the Ogataea minuta strain;and a step of disrupting a YPS1 gene in the same strain.
 25. A processof claim 24, wherein the Ogataea minuta strain is from the strain IFO10746.
 26. A process according to claim 24, which further comprises astep of disrupting at least one gene selected from the group consistingof a URA3 gene comprising the nucleotide sequence represented by SEQ IDNO:15, an ADE1 gene comprising the nucleotide sequence represented bySEQ ID NO:27, an HIS3 gene comprising the nucleotide sequencerepresented by SEQ ID NO:99, and a LEU2 gene comprising the nucleotidesequence represented by SEQ ID NO:107.
 27. A process according to claim24, which further comprises a step of disrupting at least one geneselected from the group consisting of a PEP4 gene comprising thenucleotide sequence represented by SEQ ID NO:51, a PRB1 gene comprisingthe nucleotide sequence represented by SEQ ID NO:57.
 28. A processaccording to claim 27, which further comprises a step of disrupting aKTR1 gene comprising the nucleotide sequence represented by SEQ ID NO:63and/or an MNN9 gene comprising the sequence represented by SEQ ID NO:69.29. A process according to any one of claims 24 to 28, which furthercomprises a step of introducing and expressing an α-1,2-mannosidase genefrom Aspergillus saitoi.
 30. A process according to claim 29, whereinthe α-1,2-mannosidase gene is expressed from a recombinant expressionvector comprising a gene expression cassette comprising: (a) a DNAcomprising a promoter of alcohol oxidase (AOX) gene which issubstantially represented by SEQ ID NO:79; (b) the α-1,2-mannosidasegene; and (c) a terminator of alcohol oxidase (AOX) gene which issubstantially represented by SEQ ID NO:80.
 31. A process according toclaim 24, which further comprises a step of introducing and expressing aPDI gene.
 32. A process according to claim 31, wherein the PDI gene is agene from Saccharomyces cerevisiae with the sequence found at GenBankAccession number M62815.
 33. A process according to claim 32, whereinthe PDI gene is expressed from a recombinant expression vectorcomprising a gene expression cassette comprising: (a) a DNA comprising apromoter of alcohol oxidase (AOX) gene which is substantiallyrepresented by SEQ ID NO:79; (b) the PDI gene; and (c) a terminator ofalcohol oxidase (AOX) gene which is substantially represented by SEQ IDNO:80.
 34. A process according claim 24, which further comprises a stepof introducing and expressing a heterologous gene.
 35. A processaccording to claim 34, wherein the heterologous gene is expressed from arecombinant expression vector comprising a gene expression cassettecomprising: (a) a DNA comprising a promoter of alcohol oxidase (AOX)gene which is substantially represented by SEQ ID NO:79; (b) theheterologous gene; and (c) a terminator of alcohol oxidase (AOX) genewhich is substantially represented by SEQ ID NO:80.
 36. A process forproducing a protein encoded by a heterologous gene, which comprisesculturing Ogataea minuta produced by the process of claim 34 in amedium, to obtain the protein comprising a mammalian type sugar chainencoded by the heterologous gene from the culture.
 37. A process forproducing an Ogataea minuta strain, which produces a mammalian typesugar chain represented by the following structural formula(Man₅GlcNAc₂):

wherein the process comprises the steps of: disrupting an OCH1 genecomprising the nucleotide sequence represented by SEQ ID NO:42 in anOgataea minuta strain; and disrupting a URA3 gene comprising thenucleotide sequence represented by SEQ ID NO:15 in the same strain; anddisrupting a PEP4 gene comprising the nucleotide sequence represented bySEQ ID NO:51 in the same strain; and disrupting a PRB1 gene comprisingthe nucleotide sequence represented by SEQ ID NO:57 in the same strain;and disrupting a YPS1 gene comprising the nucleotide sequencerepresented by SEQ ID NO:115 in the same strain.
 38. A process accordingto claim 37, wherein the Ogataea minuta strain is from the strain IFO10746.
 39. A process according to claim 37 or 38, which furthercomprises a step of disrupting an ADE1 gene comprising the nucleotidesequence represented by SEQ ID NO:27.
 40. A process according to claim39, which further comprises a step of disrupting a KTR1 gene comprisingthe nucleotide sequence represented by SEQ ID NO:63.
 41. A processaccording to claim 40, which further comprises a step of disrupting anHIS3 gene comprising the nucleotide sequence represented by SEQ IDNO:99.
 42. A process according to claim 40, which further comprises astep of disrupting a LEU2 gene comprising the nucleotide sequencerepresented by SEQ ID NO:107.
 43. A process according claim 37, whichfurther comprises a step of introducing and expressing anα-1,2-mannosidase gene.
 44. A process according to claim 43, wherein theα-1,2-mannosidase gene is expressed from a recombinant expression vectorcomprising a gene expression cassette comprising: (a) a DNA comprising apromoter of alcohol oxidase (AOX) gene which is substantiallyrepresented by SEQ ID NO:79; (b) the α-1,2-mannosidase gene; and (c) aterminator of alcohol oxidase (AOX) gene which is substantiallyrepresented by SEQ ID NO:80.
 45. A process according to claim 37, whichfurther comprises a step of introducing and expressing a PDI gene fromSaccharomyces cerevisiae with the sequence found at GenBank Accessionnumber (M62815).
 46. A process according to claim 45, wherein the PDIgene (M62815) is expressed from a recombinant expression vectorcomprising a gene expression cassette comprising: (a) a DNA comprising apromoter of alcohol oxidase (AOX) gene which is substantiallyrepresented by SEQ ID NO:79; (b) the PDI gene with the sequence found atGenBank Accession number M62815; and (c) a terminator of alcohol oxidase(AOX) gene which is substantially represented by SEQ ID NO:80.
 47. Aprocess according to claim 37, which further comprises a step ofintroducing and expressing a heterologous gene.
 48. A process accordingto claim 47, wherein the heterologous gene is expressed from arecombinant expression vector comprising a gene expression cassettecomprising: (a) a DNA comprising a promoter of alcohol oxidase (AOX)gene which is substantially represented by SEQ ID NO:79; (b) theheterologous gene; and (c) a terminator of alcohol oxidase (AOX) genewhich is substantially represented by SEQ ID NO:80.
 49. A process forproducing a protein encoded by a heterologous gene comprising amammalian type sugar chain, wherein the process comprises culturingOgataea minuta produced by the process of claim 47 in a medium to obtainthe protein from the culture.
 50. The process of claim 10, wherein theyeast endoplasmic reticulum (ER) retention signal has the sequence ofSEQ ID NO:
 121. 51. The process of claim 24, wherein the OCH1 gene hasthe sequence of SEQ ID NO:
 42. 52. The process of claim 24, wherein theYPS1 gene has the sequence of SEQ ID NO: 115.